Crystalline forms of piperazine-1,4-diylbis((6-(1h-benzo[d]imidazo-2-yl)pyridin-2yl)methanone)

ABSTRACT

Crystalline forms of piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone), compositions containing one or more crystalline forms of piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone), processes for preparing crystalline forms of piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone), and methods of using crystalline forms of piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

FIELD OF THE INVENTION

The present invention is directed to crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),compositions containing one or more crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),processes for preparing crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),and methods of using crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

BACKGROUND

Piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)(omilancor) is a small molecule that targets lanthionine synthetaseC-like 2 (LANCL2). LANCL2 is a receptor expressed in mammalian cellsthat has been connected to immune and metabolic responses. LANCL2 hasbeen shown to be differentially expressed in regulatory CD4+ T cells andsupports the suppressive capacity of these cells. As such, LANCL2 is atherapeutic target linked to the promotion of anti-inflammatoryresponses and restoration of immune tolerance. LANCL2 has particularimportance in the field of autoimmune and chronic inflammatory disease.The activation of LANCL2 has also been connected to improved systemicglycemic control and insulin sensitivity and altered metabolic processeswithin immune cells.

Piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has been studied in the context of inflammatory bowel disease andcolitis. When orally administered,piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)is poorly systemically absorbed, allowing for high concentrations ofdrug in the targeted local environment without generating high systemicexposures. In mice,piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)reduces local TNFα production in the gastrointestinal tract and fecalcalprotectin levels, two key biomarkers of response in IBD. In murineand human cells,piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)induces changes in CD25/STAT5 signaling and late-stage glycolysis tosupport the differentiation of Tregs and the maintenance of Tregphenotype.

Modulation of the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has the potential to improve purity, obtain reproducible particle size,and finely tune the pharmacokinetic profile to provide adequate levelsofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)to the target site for the treatment of diseases such as includingchronic inflammatory, immune-mediated, and autoimmune diseases.

There is a need for stable crystal forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

SUMMARY OF THE INVENTION

The present invention is directed to crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).The invention is also directed to compositions, including pharmaceuticalcompositions, containing one or more crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).The invention is further directed to processes for preparing crystallineforms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).The invention is further directed to methods of using the crystallineforms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),such as in the therapeutic treatment of disease.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. X-ray powder diffraction (XRPD) graph displaying a 20diffractogram ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)(BT-11) free base (Form 0).

FIG. 2. Thermogravimetry/differential thermal analysis (TG/DTA)thermogram of BT-11 free base Form 0. The thermal gravimetric trace (TG)trace and differential thermal (DT) trace are indicated.

FIG. 3. Power spectral density (PSD) graph of volume percentage versusparticle size of BT-11 free base Form 0

FIGS. 4A and 4B. XRPD 2θ diffractograms displaying BT-11 dihydrochlorideForm 1 made by Method A (FIG. 4A) and Method B (FIG. 4B).

FIG. 5. TG/DTA thermogram of BT-11 dihydrochloride Form 1. The thermalgravimetric trace (TG) trace and differential thermal (DT) trace areindicated.

FIGS. 6A-6C. Differential scanning calorimetry (DSC) thermograms ofBT-11 dihydrochloride Form 1 at a first heating step (FIG. 6A), coolingstep (FIG. 6B), and second heating step (FIG. 6C).

FIGS. 7A and 7B. Dynamic vapor sorption (DVS) isotherm (double cycle)(FIG. 7A) and DVS kinetic plot (FIG. 7B) for the analysis of BT-11dihydrochloride Form 1.

FIG. 8. Variable temperature X-ray powder diffraction (VT-XRPD) of BT-11dihydrochloride Form 1.

FIG. 9. VT-XRPD of BT-11 dihydrochloride Form 1.

FIG. 10. PSD graph of volume percentage versus particle size for BT-11dihydrochloride Form 1.

FIG. 11. XRPD 2θ diffractograms of BT-11 dihydrochloride Form 2 producedfrom Methods I, II, and III.

FIG. 12. Post-drying XRPD 2θ diffractograms of BT-11 dihydrochlorideForm 2 produced from Methods IV and V.

FIG. 13. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 2 producedupon slurrying BT-11 free base in NMP:methanol:water (51:40:9) orNMP:ethyl acetate (81:19).

FIG. 14A and FIG. 14B. XRPD diffractograms for BT-11 dihydrochlorideForm 2 produced from Methods XV, XVI, XVII, XVIII, XIX, XX, XXI, andXXII.

FIG. 15. TG/DTA thermogram of BT-11 dihydrochloride Form 2. The thermalgravimetric trace (TG) trace and differential thermal (DT) trace areindicated.

FIG. 16. TG/DTA thermogram for BT-11 dihydrochloride Form 2 producedfrom Methods XV and XIX.

FIG. 17. TG/DTA thermogram for BT-11 dihydrochloride Form 2 producedfrom Methods XVI, XVII, XVIII, XX, XXI, and XXII.

FIG. 18. DSC thermogram of BT-11 dihydrochloride Form 2.

FIG. 19A and FIG. 19B. DVS isotherm (double cycle) (FIG. 19A) and DVSkinetic plot (FIG. 19B) for the analysis of BT-11 dihydrochloride Form2.

FIG. 20. PSD graph of volume percentage versus particle size for BT-11dihydrochloride Form 2.

FIGS. 21A-21D. XRPD 2θ diffractograms of BT-11 dihydrochloride Form 3produced from various solvents.

FIG. 22. TG/DTA thermogram of BT-11 dihydrochloride Form 3. The thermalgravimetric trace (TG) trace and differential thermal (DT) trace areindicated.

FIG. 23. DSC thermogram of BT-11 dihydrochloride Form 3.

FIGS. 24A and 24B. DVS isotherm (double cycle) (FIG. 24A) and DVSkinetic plot (FIG. 24B) for the analysis of BT-11 dihydrochloride Form3.

FIG. 25. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 4 producedfrom acetic acid.

FIG. 26. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 5 producedfrom acetonitrile.

FIG. 27. TG/DTA thermogram of BT-11 dihydrochloride Form 5. The thermalgravimetric trace (TG) trace and differential thermal (DT) trace areindicated.

FIG. 28. XRPD 2θ diffractograms of BT-11 dihydrochloride Form 6 producedfrom water.

FIG. 29. TG/DTA thermogram of BT-11 dihydrochloride Form 6. The thermalgravimetric trace (TG) trace and differential thermal (DT) trace areindicated.

FIG. 30. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 7 producedupon evaporation of MeOH/water (80:20% v/v).

FIG. 31. XRPD 2θ diffractograms of BT-11 dihydrochloride Form 8 producedfrom 2-propanol.

FIG. 32. TG/DTA thermogram of the BT-11 dihydrochloride Form 8. Thethermal gravimetric trace (TG) trace and differential thermal (DT) traceare indicated.

FIG. 33. XRPD 2θ diffractograms of BT-11 dihydrochloride Form 9 producedfrom ethanol.

FIG. 34. TG/DTA thermogram of the BT-11 dihydrochloride Form 9. Thethermal gravimetric trace (TG) trace and differential thermal (DT) traceare indicated.

FIG. 35. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 10 producedupon slurrying Form 3 in pH 1.2 buffer.

FIG. 36. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 11 producedupon slurrying BT-11 free base in eight buffers.

FIG. 37. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 12 producedupon slurrying BT-11 free base in NMP:methanol (90:10).

FIG. 38. XRPD 2θ diffractogram of BT-11 dihydrochloride Form 13 producedupon slurrying BT-11 free base in dimethylsulfoxide (DMSO):methanol(50:50).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides various forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).The general term“piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)”encompasses any form thereof, including any salt, hydrate, solvate, freeacid form, free base form, crystalline form, co-crystalline form,amorphous form, or polymorph thereof. An exemplary free base form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has the following structure:

An exemplary dihydrochloride form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has the following structure:

Piperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)is al so referred to herein as “BT-11,” consistent with the currentliterature. See U.S. Pat. No. 9,556,146 to Bassaganya-Riera et al.; U.S.Pat. No. 9,839,635 to Bassaganya-Riera et al.; U.S. Pat. No. 10,028,950to Bassaganya-Riera et al.; U.S. Pat. No. 10,201,538 to Bassaganya-Rieraet al.; U.S. Pat. No. 10,493,072 to Bassaganya-Riera et al.; U.S. Pat.No. 10,682,349 to Bassaganya-Riera et al.; U.S. Pat. No. 10,849,895 toBassaganya-Riera et al; US 2019/0160100 A1 to Bassaganya-Riera et al.;Bissel et al. 2016 (Bissel P. Boes K, Hinckley J, Jortner B S,Magnin-Bissel G, Werre S R, Ehrich M, Carbo A, Philipson C, HontecillasR, Philipson N, Gandour R D, Bassaganya-Riera. J. Exploratory StudiesWith BT-11: A Proposed Orally Active Therapeutic for Crohn's Disease.Int J. Toxicol. 2016 September; 35(5):52-9); and Carbo et al. 2016(Carbo A, Gandour R D, Hontecillas R, Philipson N, Uren A,Bassaganya-Riera J. An N,N-Bis(benzimidazolylpicolinoyl)piperazine(BT-11): A Novel Lanthionine Synthetase C-Like 2-Based Therapeutic forInflammatory Bowel Disease. J Med Chem. 2016 Nov. 23;59(22):10113-10126); Leber et al. 2018 (Leber A, Hontecillas R,Zoccoli-Rodriguez V, Bassaganya-Riera J. Activation of LANCL2 by BT-11Ameliorates IBD by Supporting Regulatory T Cell Stability ThroughImmunometabolic Mechanisms. Inflamm Bowel Dis. 2018 Aug. 16;24(9):1978-1991); Leber et al. 2019 Int J Toxicol. (Leber A, HontecillasR, Zoccoli-Rodriguez V, Ehrich M, Davis J. Chauhan J, Bassaganya-RieraJ. Nonclinical Toxicology and Toxicokinetic Profile of an OralLanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11. Int J Toxicol.2019 March/April; 38(2):96-109); Leber et al. 2019 J Immunol. (Leber A,Hontecillas Zoccoli-Rodriguez V, Chauhan J, Bassaganya-Riera. J. OralTreatment with BT-11 Ameliorates Inflammatory Bowel Disease by EnhancingRegulatory T Cell Responses in the Gut. J Immunol. 2019 Apr. 1;202(7):2095-2104); and Leber et al. 2020 (Leber A, Hontecillas R,Zoccoli-Rodriguez V, Colombel Chauhan Ehrich M, Farinola N,Bassaganya-Riera J. The Safety, Tolerability, and PharmacokineticsProfile of BT-11, an Oral, Gut-Restricted Lanthionine Synthetase C-Like2 Agonist Investigational New Drug for Inflammatory Bowel Disease: ARandomized, Double-Blind, Placebo-Controlled Phase I Clinical Trial.Inflamm Bowel Dis. 2020 Mar. 4; 26(4):643-652).

The invention particularly provides various crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).Exemplary crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)provided herein include Form 0, Form 1, Form 2, Form 3, Form 4, Form 5,Form 6, Form 7, Form 8, Form 9, Form 10, Form 11, Form 12, and Form 13.

Crystalline compounds are solids with ordered arrays of molecules,whereas amorphous compounds are composed of disordered molecules. Thesearrays are also termed crystal lattices and are composed of repeatingstructural segments called unit cells. When the same molecule, such asan organic molecule, can order itself in a solid in more than one way,that molecule exhibits what is called polymorphism. For example, theelement carbon exhibits polymorphism (in elements it is termedallotropism). Solid carbon exists in at least three known crystallineforms: graphite, diamond, and fullerenes. Although each crystalline formis carbon, each has different properties because the solid-statestructure of each form differs. For example, whereas diamond is one ofthe hardest substances known, graphite is extremely soft. Many organiccompounds are also known to be polymorphic in that their structuresdiffer in how they pack together to form crystalline solids. See e.g.,Stephenson, G. A; Stowell, J. G; Toma, P. H; Dorman, D. E.; Greene, J.R.; Byrne, S. R.; “Solid state analysis of polymorphic, isomorphic andsolvated forms of Dirithromycin”, J. Am. Chem. Soc., 1994,116, 5766.

Based on a chemical structure, which is the chemical connectivity ofatoms to make a molecule, one cannot predict with any degree ofcertainty whether a compound will crystallize, under what conditions itwill crystallize, how many crystalline forms of the compound mightexist, or the solid-state structure of any of those forms. The term“solid-state structure” as used herein means the structure obtained whenmolecules pack together to form a solid.

Sometimes solvent or water molecules become incorporated into thecrystal lattice of a crystalline solid. Such a crystalline solid may bereferred to as a solvate or hydrate, respectively. Solvates, hydrates,and polymorphs are often called crystalline forms or crystalline solidforms. Here, as in most of the solid-state chemical arts, weakly boundsolvates and hydrates are also included as crystalline forms where thesolvent or water molecules are in channels or not incorporated into thecrystal lattice. Amorphous forms are often referred to as solid forms,but they are not crystalline solid forms.

Different crystalline forms of the same compound often possess differentsolid-state properties such as melting point, solubility, handling, andstability. Thus, once different crystalline forms of the same compoundhave been identified, the optimum crystalline form under any given setof processing and manufacturing conditions may be determined as well asthe different solid-state properties of each crystalline form.

There are a number of analytical methods one of ordinary skill in theart in solid-state chemistry can use to analyze solid forms. The term“analyze” as used herein means to obtain information about thesolid-state structure of solid forms. For example, X-ray powderdiffraction is a suitable technique for differentiating amorphous formsfrom crystalline forms and for characterizing and identifyingcrystalline forms of a compound. X-ray powder diffraction is alsosuitable for quantifying the amount of a crystalline form (or forms) ina mixture. In X-ray powder diffraction, X-rays are directed onto acrystal and the intensity of the diffracted X-rays is measured as afunction of twice the angle between the X-ray source and the beamdiffracted by the sample. The intensity of these diffracted X-rays canbe plotted on a graph as peaks with the x-axis being twice the angle(this is known as the “2θ” angle) between the X-ray source and thediffracted X-rays and with the y-axis being peak intensity of thediffracted X-rays. This graph is called an X-ray powder diffractionpattern or powder pattern. Different crystalline forms exhibit differentpowder patterns because the location of the peaks on the x-axis is aproperty of the solid-state structure of the crystal.

Such powder patterns, or portions thereof, can be used as an identifyingfingerprint for a crystalline form. Thus, one could take a powderpattern of an unknown sample and compare that powder pattern with areference powder pattern. A positive match would mean that the unknownsample is of the same crystalline form as that of the reference. Onecould also analyze an unknown sample containing a mixture of solid formsby adding and subtracting powder patterns of known compounds.

When selecting peaks in a powder pattern to characterize a crystallineform or when using a reference powder pattern to identify a form, oneidentifies a peak or collection of peaks in one form that are notpresent in the other solid forms.

The term “characterize” as used herein means to select an appropriateset of data capable of distinguishing one solid form from another. Thatset of data in X-ray powder diffraction is the position of one or morepeaks. Selecting whichpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)X-ray powder diffraction peaks define a particular form is said tocharacterize that form.

The term “identify” as used herein means taking a selection ofcharacteristic data for a solid form and using those data to determinewhether that form is present in a sample. In X-ray powder diffraction,those data are the x-axis positions of the one or more peakscharacterizing the form in question as discussed above. For example,once one determines that a select number of X-ray diffraction peakscharacterize a particular solid form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),one can use those peaks to determine whether that form is present in asample containingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

When characterizing and/or identifying crystalline forms of the samechemical compound with X-ray powder diffraction, it is often notnecessary to use the entire powder pattern. A smaller subset of theentire powder pattern can often be used to perform the characterizationand/or identification. By selecting a collection of peaks thatdifferentiate the crystalline form from other crystalline forms of thecompound, one can rely on those peaks to both characterize the form andto identify the form in, for example, an unknown mixture. Additionaldata can be added, such as from another analytical technique oradditional peaks from the powder pattern, to characterize and/oridentify the form.

Due to differences in instruments, samples, and sample preparation, peakvalues can be reported with the modifier “about” in front of the peakvalues. This is common practice in the solid-state chemical arts becauseof the variation inherent in peak values. A typical precision of the 2θx-axis value of a peak in a powder pattern is on the order of plus orminus 0.2° 2θ. Thus, a powder diffraction peak that appears at “about9.2° 2θ,” means that the peak could be between 9.0° 2θ and 9.4° 2θ whenmeasured on most X-ray diffractometers under most conditions.Variability in peak intensity is a result of how individual crystals areoriented in the sample container with respect to the external X-raysource (known as “preferred orientation”). This orientation effect doesnot provide structural information about the crystal.

X-ray powder diffraction is just one of several analytical techniquesone may use to characterize and/or identify crystalline forms.Spectroscopic techniques such as Raman (including microscopic Raman),infrared, and solid-state NMR spectroscopies may be used to characterizeand/or identify crystalline forms. These techniques may also be used toquantify the amount of one or more crystalline forms in a mixture.

Thermal techniques such as melting point do not necessarily, in and ofthemselves, characterize and/or identify different crystalline forms ofa compound because it is possible that different crystalline forms ofthe same compound would have indistinguishable melting points. In suchcircumstances, however, melting points could be used together withanother analytical method, such as X-ray powder diffraction, tocharacterize and/or identify crystalline forms.

The entire X-ray powder diffraction may be used to characterize eachcrystalline form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),however, one may select a smaller subset of peaks in each pattern tocharacterize each crystalline form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).Those selected peaks may then be used to identify the presence ofparticular crystalline forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in an unknown sample of or containingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

Various methods of characterizing crystalline forms are provided by U.S.Pat. Nos. 8,372,995 and 10,537,565, each of which is incorporated hereinby reference.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, or each 2θ value range(s) selected from 12.4to 12.8, 16.9 to 17.3, 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6, and28.2 to 28.6 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation. In some versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within one or both 2θ value range(s) selected from 12.4 to12.8 and 16.9 to 17.3. In some versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 12.4 to 12.8 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 16.9 to 17.3 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, at least three, or each 2θvalue range(s) selected from 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6,and 28.2 to 28.6 degrees. In some versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 9.8 to 10.2 degrees. Insome versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 14.1 to 14.5 degrees. Insome versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 19.2 to 19.6 degrees. Insome versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 25.3 to 25.7 degrees. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 2.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 11, 12, 13, 14A, and 14B. “Substantially similar” in this contextand other similar contexts herein refers to an X-ray powder diffractionpattern having peaks at about the same 2θ values as the peaks present inthe patterns provided herein, wherein “about” refers to a value of plusor minus 0.2° 2θ. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 2.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 5. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 2.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=12.81 Å, α=90°, b=12.56 Å, β=105.25°,c=9.48 Å, γ=90°. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 2.

Form 2 ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has many advantages over the other forms provided herein. Theseadvantages include increased thermostability, a substantially normalizedand uniform size distribution (compare, e.g., FIG. 20 with FIGS. 3 and10), and enhanced gut-restricted pharmacokinetic properties after oraladministration (see, e.g., Table 20). The increased thermostabilityresults in an increased shelf-life. The normalized and uniform size isadvantageous for formulatingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in compositions, such as pharmaceutical compositions. In particular,control of particle size improves batch-to-batch consistency in tissuepenetration and bioavailability, potentially improving reproducibilityof clinical efficacy and performance in addition to normalizingmanufacturing processes. The enhanced gut-restricted pharmacokineticproperties are advantageous for orally administeringpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)for the treatment of intestinal diseases, such as inflammatory boweldiseases (e.g., ulcerative colitis and/or Crohn's disease).

Any aspect of the invention described herein as pertaining to a crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)exemplified by Form 2 can have a Dv50 within a range of about 13-55 μm,such as about 20-48 μm, about 27-41 μm or about 26.9-40.7 μm. Any aspectof the invention described herein as pertaining to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)exemplified by Form 2 can have a Dv10 within a range of about 5-28 μm,such as about 9-24, about 13-20 μm, or about 12.5-20.3 μm. Any aspect ofthe invention described herein as pertaining to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)exemplified by Form 2 can have a Dv90 within a range of about 18-117 μm,such as about 34-100, about 51-84, or about 50.9-83.9 μm. Any aspect ofthe invention described herein as pertaining to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)exemplified by Form 2 can have a Dv50 within a range of about 13-55 μm,such as about 20-48 μm, about 27-41 μm or about 26.9-40.7 μm; a Dv10within a range of about 5-28 μm, such as about 9-24, about 13-20 μm, orabout 12.5-20.3 μm; and a Dv90 within a range of about 18-117 μm, suchas about 34-100, about 51-84, or about 50.9-83.9 μm, with the provisothat the Dv10 is a lower value than the Dv50 and the Dv50 is a lowervalue than the Dv90. “Dv50,” “Dv10,” and “Dv90” are common terms in theart pertaining to particle size and refer to the maximum particle sizefor a given percentage volume of a sample, wherein “D” refers todiameter, “v” refers to a distribution weighting by volume, and “50,”“10,” and “90” refer to percentages of sample below the given diametervalues. For example, the Dv50 is the maximum particle diameter belowwhich 50% of the same volume exists. The Dv50 is also known as themedian particle size by volume. The Dv50, the Dv10, and the Dv90together provide an indication of the width or breadth of a particlesize distribution.

An aspect of the invention is a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, at least six, or each 2θ value range(s)selected from 10.1 to 10.5, 13.8 to 14.2, 16.9 to 17.3, 23.7 to 24.1,24.3 to 24.7, 27.7 to 28.1, and 28.8 to 29.2 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation. In someversions, thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, at least three, or each 2θvalue range(s) selected from 10.1 to 10.5, 16.9 to 17.3, 23.7 to 24.1,and 28.8 to 29.2 degrees. In some versions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 10.1 to 10.5 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 16.9 to 17.3 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 23.7 to 24.1 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 28.8 to 29.2 degrees. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, or each 2θ value range(s)selected from 13.8 to 14.2, 24.3 to 24.7, and 27.7 to 28.1 degrees. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 3.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 21A, 21B, 21C, and 21D. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 3.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=9.30 Å, α=71.15°, b=11.78 Å, β=106.99°,c=10.20 Å, γ=108.35°. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 3.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 7. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 3.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, at least six, or each 2θ value range(s)selected from 5.7 to 6.1, 9.6 to 10.0, 14.0 to 14.4, 19.4 to 19.8, 23.0to 23.4, 24.1 to 24.5, and 27.9 to 28.3 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation. In someversions, the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 5.7 to 6.1. An exemplary crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 1.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 4A and 4B. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 1.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 3. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 1.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=6.97 Å, α=98.26°, b=15.17 Å, β=101.74°,c=9.31 Å, γ=89.23°. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 1.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, or each 2θ value range(s) selected from 12.0to 12.4, 15.0 to 15.4, 15.3 to 15.7, 21.9 to 22.3, 22.2 to 22.6, and28.0 to 28.4 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 0.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 1. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 0.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 1. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 0.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 6.9 to 7.3, 12.9 to 13.3, 23.4 to 23.8,and 27.3 to 27.7 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 4.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 25. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 4.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 9. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 4.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 3.5 to 3.9, 7.2 to 7.6, 14.6 to 15.0,and 24.5 to 24.9 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 5.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 26. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 5.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 10. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 5.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, or each 2θ valuerange(s) selected from 9.2 to 9.6, 26.6 to 27.0, and 27.6 to 28.0degrees in an X-ray powder diffraction pattern obtained using Cu K alpharadiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 6.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 28. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 6.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 11. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 6.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 9.9 to 10.3, 12.1 to12.5, 20.5 to 20.9, 25.7 to 26.1, and 26.9 to 27.3 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 7.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 30. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 7.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 13. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 7.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 7.4 to 7.8, 13.1 to13.5, 22.1 to 22.5, 23.9 to 24.3, and 25.1 to 25.5 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 8.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 31. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 8.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 15. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 8.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 9.9 to 10.3, 12.1 to 12.5, 25.8 to 26.2,and 26.9 to 27.3 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 9.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 33. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 9.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 16. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 9.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, or each 2θ value range(s) selected from 8.8to 9.2, 9.4 to 9.8, 19.1 to 19.5, 23.0 to 23.4, 26.0 to 26.4, and 27.3to 27.7 degrees in an X-ray powder diffraction pattern obtained using CuK alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 10.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 35. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 10.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction peak at about any one or more 2θvalues provided in Table 18. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 10.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 15.6 to 16.0, 19.4to 19.8, 21.4 to 21.8, 23.3 to 23.7, and 27.1 to 27.5 degrees in anX-ray powder diffraction pattern obtained using Cu K alpha radiation. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 11.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in FIG. 36. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 11.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 11.6 to 12.0, 18.3 to 18.7, 27.1 to27.5, 28.0 to 28.4 degrees in an X-ray powder diffraction patternobtained using Cu K alpha radiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 12.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 37. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 12.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, or each 2θ valuerange(s) selected from 9.4 to 9.8, 17.0 to 17.4, and 24.5 to 24.9degrees in an X-ray powder diffraction pattern obtained using Cu K alpharadiation. An exemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 13.

An aspect of the invention is directed to a crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 38. Anexemplary crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having these characteristics is provided herein as Form 13.

The invention further provides processes for preparing crystal forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).The methods generally comprise mixingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in a solvent to form a slurry. The methods can further compriseisolating crystallized material from the slurry to obtain isolatedmaterial. The methods can further comprise washing the isolated materialwith a solvent to obtain washed material. The methods can furthercomprise drying either the isolated material or the washed material toobtain dried material comprising the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

For the preparation of Form 2, the solvent can comprise at least one ofmethanol, 2-ethoxyethanol, methyl isobutyl ketone, N-methylpyrrolidone,and dimethylsulfoxide. In some versions, the solvent is a mixture ofdimethylsulfoxide, methanol, and water. In some versions, the mixture ofdimethylsulfoxide, methanol, and water is in a ratio of 45-55:35-45:5-15(dimethylsulfoxide:methanol:water), such as 50:40:10. In some versions,the solvent is N-methylpyrrolidone or a mixture of N-methylpyrrolidoneand one or more of methanol, ethanol, 2-propanol, 1-propanol,acetonitrile, acetone, ethyl acetate, and water. In some versions, thesolvent is selected from the group consisting of 2-ethoxyethanol, methylisobutyl ketone, or a mixture of methanol and water. Some versionscomprise isolating crystallized material from the slurry to obtainisolated material; washing the isolated material with a solventcomprising methanol or N-methylpyrrolidone to obtain washed material;and drying the washed material to obtain dried material comprising thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).Some versions further comprise seeding the slurry withpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)Form 2. Some versions further comprise cyclically heating and coolingthe slurry.

In some versions, the preparation of Form 2 comprises addingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)to DMSO:methanol:water to form a slurry. In some versions, theDMSO:methanol:water is in a ratio of 45-55:35-45:5-15(dimethylsulfoxide:methanol:water), or about 50:40:10. In some versions,thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)is added to the DMSO:methanol:water in a 1:15-1:50 (w/v) ratio, such asa 1:33 (w/v) ratio, to form the slurry. In some versions, the mixture ofthepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in the DMSO:methanol:water is stirred stirring at 25-35° C. for a timeperiod to form the slurry. In some versions, the time period isapproximately 5-15 minutes, such as approximately 10 minutes. In someversions, the temperature of the slurry is then raised. In someversions, the temperature of the slurry is raised to 50-75° C., such as60-65° C. In some versions, approximately 2 to 3 equivalents of HCl isthen added to the slurry over a time period. In some versions, the addedHCl is in DMSO:methanol:water, such as in a ratio of 45-55:35-45:5-15(dimethylsulfoxide:methanol:water), or about 50:40:10. In some versions,the time period in which the HCl is added is about 1.5-2.5 hours. Insome versions, the reaction is then filtered to form a filtrate. In someversions, the filtering occurs at a temperature 60-65° C. In someversions, Form 2 ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)is added to the filtrate to form a second slurry. The Form 2 can beadded in an amount of about 1 to 2 percent weight of the filtrate. Insome versions, the second slurry is stirred for a time period. In someversions, the time period is about 6-8 hours. In some versions, thestirring occurs at 60-65° C. In some versions, the second slurry is thencooled. In some versions, the second slurry is cooled to about 20-30° C.In some versions, the second slurry is cooled at a rate of approximately8° C./hour. In some versions, the second slurry is then stirred at 20 to30° C. for 1 to 2 hours. In some versions, the second slurry is thenfurther cooled. In some versions, the further cooling is to atemperature of 0 to 5° C. at a rate of approximately 6° C./hour. In someversions, the second slurry is then stirred at 0 to 5° C. for 12 to 14hours. In some versions, solid material is then filtered, and wetmaterial is slurried in methanol:water. The wet material can be slurriedfor 5 to 96 hours at 25 to 55° C. In some versions, the wet slurriedmaterial is filtered to obtain a filtrate. In some versions, thefiltrate is dried. In some versions, the filtrate is dried under vacuumat 25 to 55° C.

For the preparation of Form 3, the solvent can comprise at least one oftetrahydrofuran, 1,4-dioxane, 1-butanol, 1-propanol, 2-methyl THF, butylacetate, dichloromethane, ethyl acetate, isopropyl alcohol, methanol,methyl ethyl ketone, and tert-butyl methyl ether.

The invention also provides compositions comprising any one or more ofthe crystal forms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of the invention. In some versions, the compositions comprisepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in any one or more crystal forms of as characterized anywhere herein inamount of at least 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w,35% w/w, 40% w/w, 45% w/w, 50% w/w, 55% w/w, 60% w/w, 65% w/w, 70% w/w,75% w/w, 80% w/w, 81% w/w, 82% w/w, 83% w/w, 84% w/w, 85% w/w, 86% w/w,87% w/w, 88% w/w, 89% w/w, 90% w/w, 91% w/w, 92% w/w, 93% w/w, 94% w/w,95% w/w, 96% w/w, 97% w/w, 98% w/w, 99% w/w, or 100% w/w of the totalamount ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)present in the composition. In some versions, the compositions comprisepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in any one or more crystal forms of as characterized anywhere herein inamount of at least 5% w/w, 10% w/w, 15% w/w, 20% w/w, 25% w/w, 30% w/w,35% w/w, 40% w/w, 45% w/w, 50% w/w, 55% w/w, 60% w/w, 65% w/w, 70% w/w,75% w/w, 80% w/w, 81% w/w, 82% w/w, 83% w/w, 84% w/w, 85% w/w, 86% w/w,87% w/w, 88% w/w, 89% w/w, 90% w/w, 91% w/w, 92% w/w, 93% w/w, 94% w/w,95% w/w, 96% w/w, 97% w/w, 98% w/w, 99% w/w, or 100% w/w of the totalamount of the crystalizedpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)present in the composition. “Crystalizedpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)”in this context refers to thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)that is not in an amorphous form.

Methods of determining the relative weight percentage of a particularcrystalline form may include comparative analysis of a sample of apiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)relative to a standard curve by x-ray powder diffraction. Samples ofknown ratios may be created by isolating purified crystalline forms andblending in ratios of 1:9, 3:7, 1:1, 7:3, and 9:1. Samples of knownratios would then be analyzed by XRPD to determine relative peak heightsor peak areas at characteristic positions for each crystalline form. Thepeak height or peak area would linearly or nonlinearly correspond to therelative weight proportion of a given crystalline form in the overallsample.

In some versions the composition is a pharmaceutical composition. Thepharmaceutical compositions may comprise any one or more of the crystalforms ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of the invention in combination or association with a pharmaceuticallyacceptable carrier. Such compositions can be used to deliverpharmaceutically effective amounts of one or more of thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)crystal forms of the invention.

The carrier can comprise any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants, and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E, W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials that can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose, starches such as cornstarch and potato starch, cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate, powderedtragacanth, malt, gelatin, talc, excipients such as cocoa butter andsuppository waxes, oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil, glycols such aspropylene glycol and polyethylene glycol, esters such as ethyl oleateand ethyl laurate, agar, buffering agents such as magnesium hydroxideand aluminum hydroxide, alginic acid, pyrogen-free water, isotonicsaline, Ringer's solution, ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives, and antioxidants. Additional carriersinclude those described in U.S. Pat. No. 9,556,146 to Bassaganya-Rieraet al.; U.S. Pat. No. 9,839,635 to Bassaganya-Riera et al.; U.S. Pat.No. 10,028,950 to Bassaganya-Riera et al.; U.S. Pat. No. 10,201,538 toBassaganya-Riera et al.; U.S. Pat. No. 10,493,072 to Bassaganya-Riera etal.; U.S. Pat. No. 10,682,349 to Bassaganya-Riera et al.; U.S. Pat. No.10,849,895 to Bassaganya-Riera et al; and US 2019/0160100 A1 toBassaganya-Riera et al.

The pharmaceutical compositions can be used to deliver pharmaceuticallyeffective amounts of one or more of thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)crystal forms of the invention to a subject. The subject can be asubject suffering from a chronic inflammatory, immune-mediated, orautoimmune disease. The crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)can be administered in an amount effective to treat the disease. Thedisease can comprise any disease or condition described in U.S. Pat. No.9,556,146 to Bassaganya-Riera et al.; U.S. Pat. No. 9,839,635 toBassaganya-Riera et al.; U.S. Pat. No. 10,028,950 to Bassaganya-Riera etal.; U.S. Pat. No. 10,201,538 to Bassaganya-Riera et al.; U.S. Pat. No.10,493,072 to Bassaganya-Riera et al.; U.S. Pat. No. 10,682,349 toBassaganya-Riera et al.; U.S. Pat. No. 10,849,895 to Bassaganya-Riera etal; and US 2019/0160100 A1 to Bassaganya-Riera et al. Examples includeinflammatory bowel disease, such as ulcerative colitis and Crohn'sdisease. The route of administration can comprise any route described inU.S. Pat. No. 9,556,146 to Bassaganya-Riera et al.; U.S. Pat. No.9,839,635 to Bassaganya-Riera et al.; U.S. Pat. No. 10,028,950 toBassaganya-Riera et al.; U.S. Pat. No. 10,201,538 to Bassaganya-Riera etal.; U.S. Pat. No. 10,493,072 to Bassaganya-Riera et al.; U.S. Pat. No.10,682,349 to Bassaganya-Riera et al.; U.S. Pat. No. 10,849,895 toBassaganya-Riera et al; and US 2019/0160100 A1 to Bassaganya-Riera etal. The disease can also or alternatively comprise an inflammatory,immune-mediated, or autoimmune condition of a surface tissue such as theskin or mucosa (e.g., oral mucosa). Exemplary diseases includepsoriasis, cutaneous lupus erythematosus, dermatomyositis, pemphigoid,pemphigus, scleroderma, vasculitis, epidermolysis bullosa acquisita,vitiligo, lichen planus, scleritis, dermatitis or eczema, erythemanodosum, pyoderma gangrenosum, skin fissures, acne, enterocutaneousfistula, skin tags, canker sores, acrodermatitis enteropathica, pyodermavegetans, leukocytoclastic vasculitis, anal fissures, Sweet's syndrome,rosacea, alopecia, keratoderma blennorrhagica, rosacea, cold sores,urticaria, actinic keratosis, carbuncle, cellulitis, ichthyosisvulgaris, skin infection, malar rash, photosensitivity, livedoreticularis, livedo reticularis, oral and nasal ulcers, purpura,mucositis, hemorrhoids, burn, and sunburn. Administration in such casescan comprise topically administering the composition to a subject.

Form 2 ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a characteristic of having an increased confinement to thegastrointestinal tract after oral administration. Form 2 is therefore apreferred form for orally administering to a subject for the treatmentof a disease of the gastrointestinal tract, such as a chronicinflammatory, immune-mediated, or autoimmune disease of thegastrointestinal tract. Exemplary chronic inflammatory, immune-mediated,or autoimmune disease of the gastrointestinal tract include inflammatorybowel disease, such as ulcerative colitis and Crohn's disease, andeosinophilic disorders of the gastrointestinal tract, such aseosinophilic esophagitis. The pharmaceutical composition comprising Form2 can be administered in an amount effective to treat the disease of thegastrointestinal tract.

The terms “crystal form” and “crystalline form” are used interchangeablyherein.

“2θ value” and grammatical variants thereof refer to values that wouldcorrespond to position 2θ values as provided herein.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

EXAMPLES Characterization Methods X-Ray Powder Diffraction (XRPD)

XRPD analysis was carried out, scanning the samples between 3 and 35°2θ. The material was gently ground to release any agglomerates andloaded onto a multi-well plate with polymer film to support the sample.The multi-well plate was then placed into the diffractometer andanalyzed using Cu K radiation (α1λ=1.54060 Å; α2=1.54443 Å; β=1.39225 Å;α1:α2 ratio=0.5) running in transmission mode (step size 0.0130° 2θ,step time 18.87 s) using 40 kV/40 mA generator settings. Data werevisualized and analyzed.

Polarized Light Microscopy (PLM)

The presence of crystallinity (birefringence) was determined using apolarizing microscope, equipped with a camera and image capturesoftware. All images were recorded using at 200× magnification using a20× objective, unless otherwise stated.

Thermogravimetric Analysis (TGA)

Approximately, 5 mg of material was weighed into an open aluminum panand loaded into a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) and held at room temperature. The sample was thenheated at a rate of 10° C./min from 20° C. to 350° C. during which timethe change in sample weight was recorded along with any differentialthermal events (DTA). Nitrogen was used as the purge gas, at a flow rateof 300 cm³/min.

Differential Scanning calorimetry (DSC)

Approximately, 5 mg of material was weighed into an aluminum DSC pan andsealed non-hermetically with a pierced aluminum lid. The sample pan wasthen loaded into a calorimeter, cooled, and held at 20° C. Once a stableheat-flow response was obtained, the sample and reference were heated tomelting (if possible) at a scan rate of 10° C./min and the resultingheat flow response monitored. Nitrogen was used as the purge gas, at aflow rate of 50 cm³/min.

Dynamic Vapor Sorption (DVS)

Approximately, 10 mg of sample was placed into a mesh vapor sorptionbalance pan and loaded into a DVS-1/DVS Intrinsic/DVS Advantage dynamicvapor sorption balance by Surface Measurement Systems. The sample wassubjected to a ramping profile from 40-90% relative humidity (RH) at 10%increments, maintaining the sample at each step until a stable weighthad been achieved (dm/dt 0.004%, minimum step length 30 min, maximumstep length 500 min) at 25° C. After completion of the sorption cycle,the sample was dried using the same procedure to 0% RH and then a secondsorption cycle back to 40% RH. Two cycles were performed. The weightchange during the sorption/desorption cycles were plotted, allowing forthe hygroscopic nature of the sample to be determined. XRPD analysis wasthen carried out on any solid retained.

Gravimetric Vapor Sorption (GVS)

Approximately 10-20 mg of sample was placed into a mesh vapor sorptionbalance pan and loaded into a sorption analyzer balance. The sample wassubjected to a ramping profile from 40-90% relative humidity (RH) at 10%increments, maintaining the sample at each step until a stable weighthad been achieved (98% step completion, minimum step length 30 minutes,maximum step length 60 minutes) at 25° C. After completion of thesorption cycle, the sample was dried using the same procedure to 0% RH,and finally taken back to the starting point of 40% RH. Two cycles wereperformed. The weight change during the sorption/desorption cycles wereplotted, allowing for the hygroscopic nature of the sample to bedetermined.

Variable Temperature X-Ray Powder Diffraction (VT-XRPD)

VT-XRPD analysis was carried out on a diffractometer equipped with atemperature chamber. The samples were scanned between 4 and 35° 2θ usingCu K radiation (α₁λ=1.54060 Å; α2=1.54443 Å; β=1.39225 Å; α₁:α₂ratio=0.5) running in Bragg-Brentano geometry (step size 0.008° 2θ)using 40 kV/40 mA generator settings.

Particle Size Distribution

Particle size distribution analysis was carried out using a particlesize analyzer with a dispersion cell. Approximately 50 mg of sample wasweighed into a 20 mL scintillation vial and 10 mL of dispersant wasadded. The sample was sonicated for 30 seconds, aspirated with a glasspipette, and then added to the dispersion unit. The sample wascirculated for 30 seconds to allow the dispersion to stabilize.

Example 1. Crystal Form 0 of BT-11 Form 0 Preparation Methods

BT-11 free base was made as described in U.S. Pat. No. 10,028,950. Asolution of 6-(1H-Benzimidazol-2-yl)pyridine-2-carboxylic acid (12 g) inDMF (100 mL) was cooled to 0° C., and then sequentially added EDC.HCl(1.5 eq), HOBt (1.5 eq) and DIPEA (1.2 eq, taken in volumes with densitypresumed). The mixture was stirred for 10 min at 0° C. Piperazine (0.5eq) was added and the reaction mixture was allowed to warm to RTgradually and stirred for 16 h. After completion of the reaction(monitored by TLC, eluent: 10% MeOH in DCM), the reaction mixture waspoured into ice-cold water (˜300 mL), the precipitated solid wasfiltered, washed with ice-cold water and dried to get BT-11 (10 g, 75%)as pale brown solid. ¹H NMR (400 MHz, DMSO-d₆), δ 13.0 (s, 1H), 12.8 (s,1H), 8.38 (dd, 2H), 8.13 (dt, 2H), 7.73 (dd, 2H), 7.67 (d, 2H), 7.57(dd, 2H), 7.25 (m, 4H), 3.90 (bs, 2H), 3.80 (bdd, 2H), 3.65 (bdd, 2H),3.56 (bs, 2H). LCMS-ES 529.44 [M+H]⁺, 265.46 [(M+2H)/2]⁺⁺.

Characterization

BT-11 free base was characterized via XRPD (FIG. 1). The free base wasfound to be crystalline. This was designated as Form 0 of BT-11.Indexing of Form 0 found the most probable space group to be P2₁/cmonoclinic. Diffraction peaks are presented in Table 1. Reduced cellparameters are presented in Table 2.

TABLE 1 Diffraction peaks observed for Form 0 of BT-11. Pos. d-spacingHeight Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts] [cts *°2θ] [%]h k l [°2θ] 1 9.4457 9.36336 329 24.91 10.43 0 2 0 9.0246 2 9.85338.97689 382.36 38.6 12.12 1 1 −1 9.869 3 12.1867 7.26278 2204.02 250.3169.84 2 0 0 12.4464 4 13.273 6.67074 495.74 43.79 15.71 2 1 0 13.244 514.3626 6.16704 458.7 40.52 14.54 2 1 −1 14.0268 6 15.1595 5.844611339.35 135.21 42.44 1 0 −2 15.072 7 15.5525 5.69779 3155.58 318.55 1000 1 2 15.5991 8 16.0682 5.51606 355.8 44.9 11.28 2 2 −1 16.0754 918.0954 4.9024 372.9 47.05 11.82 0 4 0 18.1059 10 18.661 4.75509 430.4148.88 13.64 3 0 0 18.7161 11 20.0651 4.4254 1003.62 126.64 31.8 1 4 −120.1522 12 22.0855 4.02491 1317.64 166.27 41.76 2 4 0 22.0309 13 22.35273.9774 2340.55 295.35 74.17 2 3 −2 22.2811 14 22.6134 3.93214 940.8271.23 29.81 1 1 −3 22.6404 15 23.1978 3.83437 329.57 41.59 10.44 3 3 023.1808 16 25.2815 3.52287 725.39 45.77 22.99 2 3 2 25.2468 17 25.92993.43624 269.83 47.67 8.55 2 5 0 25.9562 18 26.3756 3.37918 298.59 30.149.46 2 5 −1 26.3747 19 28.1953 3.16509 1136.22 143.38 36.01 3 4 128.1945 20 31.0896 2.87672 766.25 87.02 24.28 4 4 0 31.0769

TABLE 2 Reduced cell parameters of Form 0 of BT-11. Cell ParametersValue a sigma [Å] 14.46 b sigma [Å] 19.58 c sigma [Å] 12.073 alpha(sigma) [°] 90.00 beta (sigma) [°] 100.67 gamma (sigma) [°] 90.00

Form 0 of BT-11 was characterized via thermogravimetric analysis (FIG.2). The thermal gravimetric trace (TG) showed consecutive losses in massfrom the outset of heating up to ˜160° C. Additionally, there was a 4.1%mass loss of approximately 1.3 equiv. of water (broad endothermic event(onset 57° C.) associated with this loss in mass. There was a 4.8% massloss above 60° C., which may be a result of dehydration (1.5 equiv. ofwater). This is indicative of a broad endothermic event (onset 136° C.)associated with this second loss in mass. An exothermic event (onset208° C.) followed by a sharp endothermic event (onset 328° C.) occurred.PSD analysis (FIG. 3) showed that Form 0 had a d₉₀ of 19.8 μm, d₅₀ of6.3 μm and d₁₀ of 0.2 μm.

Example 2. Crystal Form 1 of BT-11 Form 1 Preparation Methods

Method A. BT-11 dihydrochloride was made as described in U.S. Pat. No.10,028,950. A suspension of BT-11 free base (1.0 eq) in minimal amountof MeOH (5 mL) was cooled to 0° C., was added 4 M methanolic HCl(excess, 15 mL/1 g) dropwise over a period of 15-20 min. The mixture wasallowed gradually to warm to RT for 3 h. After completion of thereaction (monitored by TLC, eluent: 10% MeOH in CH₂Cl₂), the volatileswere evaporated under reduced pressure. The crude material was washedwith 10% MeOH in CH₂Cl₂ and lyophilized to get an off-white solid (850mg, 75%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.58 (dd, 2H), 8.29 (dt, 2H),7.83 (m, 6H), 7.44 (bd, 4H), 3.91 (bs, 2H), 3.81 (bm, 2H), 3.64 (bm,2H), 3.55 (bs, 2H). LCMS-ES 529.56 [M+H]⁺.

Method B. A suspension of BT-11 free base in minimal ethylacetate wascooled to −5 to 0 C. 4M HCl in ethylacetate was added over a period of25 to 30 minutes. Reaction mass was maintained at −5 to 5 C for 4 to 5hours. After completion of the reaction, the wet material was washedwith ethylacetate and dried under vacuum. ¹H NMR (400 MHz, DMSO-d₆), δ8.58 (dd, 2H), 8.29 (dt, 2H), 7.83 (m, 6H), 7.44 (bd, 4H), 3.91 (bs,2H), 3.81 (bm, 2H), 3.64 (bm, 2H), 3.55 (bs, 2H). LCMS-ES 529.56 [M+H]⁺.

Characterization

The BT-11 dihydrochloride preparations were characterized via XRPD(FIGS. 4A and 4B). The material was found to be crystalline anddesignated as Form 1. Diffraction peaks are presented in Table 3.Reduced cell parameters are presented in Table 4.

TABLE 3 Diffraction peaks observed for Form 1 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 5.8988 14.98305 685.76 77.88 46.89 0 1 05.8829 2 9.7766 9.04711 980.73 111.38 67.06 0 0 1 9.796 3 10.65428.30379 478.95 48.35 32.75 0 1 −1 10.6804 4 11.7931 7.5043 434.36 38.3729.7 0 2 0 11.7814 5 14.1862 6.24333 628.01 63.4 42.94 1 −1 0 14.1623 615.6073 5.67788 148.17 29.92 10.13 1 1 −1 15.2801 7 16.4538 5.38765173.48 21.89 11.86 0 2 1 16.4013 8 17.763 4.99338 174.9 35.31 11.96 1 01 17.8131 9 18.9757 4.67692 537.95 81.46 36.78 0 3 −1 18.9875 10 19.61124.52678 962.24 85 65.79 0 0 2 19.6645 11 20.6095 4.30971 153.99 31.0910.53 1 −2 1 20.4799 12 21.5298 4.12752 170.69 25.85 11.67 0 3 1 21.494713 22.2248 4 687.43 69.4 47 1 3 0 22.1861 14 23.1555 3.84129 1462.49129.18 100 1 2 −2 23.0512 15 24.3124 3.66105 1438.67 127.08 98.37 0 4 −124.3218 16 26.0833 3.41638 151.74 38.29 10.38 2 0 0 26.1062 17 28.12093.17329 728.59 165.49 49.82 2 2 −1 28.1666 18 28.6645 3.11433 211.9132.09 14.49 0 4 −2 28.646 19 31.0084 2.88407 306.81 46.46 20.98 2 3 −131.0171 20 33.2962 2.69095 146.15 36.88 9.99 0 5 −2 33.3586

TABLE 4 Reduced cell parameters of Form 1 of BT-11 dihydrochloride. CellParameters Value a sigma [Å] 6.97 b sigma [Å] 15.17 c sigma [Å] 9.31alpha (sigma) [°] 98.26 beta (sigma) [°] 101.74 gamma (sigma) [°] 89.23

BT-11 Form 1 was characterized via PLM. The PLM images showed that thematerial consisted of aggregated particles with no clear morphology.Additionally, the material appeared birefringent under polarized light,indicative of a crystalline material.

BT-11 Form 1 was characterized via thermal gravimetric trace (FIG. 5).The thermal gravimetric trace showed consecutive losses in mass observedfrom the outset of heating up to ˜220° C. Weak endothermic events wereobserved (onsets of ˜84 and 120° C., respectively) around these initialmass losses. The differential thermogram (TG) showed a weak exothermicevent (onset 250° C.) followed by two endothermic events (onsets of 287and 311° C.). No further loss in mass until second endothermic event.

BT-11 Form 1 was characterized via DSC (FIGS. 6A-6C). The material washeated in the DSC to around 130° C. The material was then cooled andre-heated to check for recrystallization. In FIG. 6A at the first heat(20-130° C.), two broad endothermic events observed (around ˜97° C. andan onset of ˜124° C., respectively) which correlated with the two weakendothermic events observed in the TG/DTA. In FIG. 6B at the coolingstep (130-20° C.), no thermal events were observed on cooling. In FIG.6C at the second heat (20-130° C.), no thermal events were observed onre-heating.

BT-11 Form 1 was characterized via DVS (FIG. 7A and FIG. 7B). In FIG.7A, DVS analysis showed a total 24% mass increase up to 90% RH. Thematerial was hygroscopic, 6.0% of total mass was lost below 20% RH.Rehydration appeared slower than dehydration. In FIG. 7B, no evidence ofre-crystallization or form change occurring during the DVS experiment.Dehydration and re-hydration steps were not very pronounced.

BT-11 Form 1 was characterized by VT-XRPD analysis (FIG. 8 and FIG. 9).In FIG. 8, several new patterns were observed during the VT-XRPDexperiment (each represented by a different color). After each mass lossor at each thermal event observed in the TG/DTA, a new (poorlycrystalline) pattern was produced. The material appeared amorphous afterthe possible salt disproportionation.

In FIG. 9, a new pattern was produced, above 250° C., where the materialhad potentially fully disproportionated and an exothermic event wasnoted. Post-degradation, a poorly crystalline pattern was observed butwas found to be predominantly amorphous when cooled back to 25° C.

BT-11 Form 1 was characterized by PSD. PSD analysis (FIG. 10) showedthat the received material had a d₉₀ of 40.8 μm, d₅₀ of 0.5 μm and d₁₀of 0.1 μm.

Form 1 was also obtained from dried material from a toluene slurry.Toluene was added to BT-11 dihydrochloride in a 100:1 (v/w) ratio toform a slurry. Slurry was thermally cycled with agitation for 72 hoursbetween ambient temperature for 4 hours followed by 40° C. for 4 hours.Solid material was isolated by filtration and dried at 40° C. for 2hours.

Example 3. Crystal Form 2 of BT-11 Form 2 Preparation Methods

Method I. Methanol:water (80:20) was added to BT-11 dihydrochloride in a40:1 (v/w) ratio to form a slurry. Slurry was then thermally cycled withagitation for 72 hours between ambient temperature for 4 hours followedby 40° C. for 4 hours. Solid material was isolated by filtration anddried at 40° C. for 2 hours. Dried material was analyzed by XRPD to havea unique crystalline form, designated to be Form 2.

Method II. 2-ethoxyethanol was added to BT-11 dihydrochloride in a 100:1(v/w) ratio to form a slurry. Slurry was then thermally cycled withagitation for 72 hours between ambient temperature for 4 hours followedby 40° C. for 4 hours. Solid material was isolated by filtration anddried at 40° C. for 2 hours. Dried material was analyzed by XRPD to havea unique crystalline form, designated to be Form 2.

Method III. Methyl isobutyl ketone was added to BT-11 dihydrochloride ina 100:1 (v/w) ratio to form a slurry. Slurry was then thermally cycledwith agitation for 72 hours between ambient temperature for 4 hoursfollowed by 40° C. for 4 hours. Solid material was isolated byfiltration and dried at 40° C. for 2 hours. Dried material was analyzedby XRPD to have a unique crystalline form, designated to be Form 2.

Method IV. BT-11 dihydrochloride was added to methanol:water (80:20) ina 1:40 (w/v) or 1:16 (w/v) ratio to form slurries under stirring at 20°C. Slurries were temperature cycled between 20° C. and 50° C. for 48hours, at a rate of 0.1° C./min with a hold of 4 hours at eachtemperature. Solid material was isolated by filtration and dried at 40°C. under vacuum for 65 hours. Dried material was analyzed by XRPD tohave a unique crystalline form, designated to be Form 2.

Method V. Methanol:water (80:20) was added to BT-11 dihydrochloride in a1:20 (w/v) ratio to form slurries under stirring at 22° C. Slurries weretemperature cycled between 30° C. and 50° C. for 48 hours, at a rate of0.1° C./min with a hold of 4 hours at each temperature. Solid materialwas isolated by filtration and dried at 40° C. under vacuum for 20hours. Dried material was analyzed by XRPD to have a unique crystallineform, designated to be Form 2.

Method VI. N-methylpyrrolidone (NMP) or a 90:10 (v/v) mixture ofNMP:methanol were added to BT-11 free base in 24:1 or 32:1 (v/w) ratios,respectively, to form slurries by stirring for 1 hour at 50° C. Thetemperature was decreased to 40° C., over approximately 10 minutes.BT-11 dihydrochloride Form 2 was seeded into the mixture (6% weight).0.55 equivalents of HCl were added dropwise. After approximately 5minutes, an additional 2% weight of BT-11 dihydrochloride Form 2 wasadded. The slurries were stirred at 40° C. for approximately 10 minutes.1.65 equivalents of HCl were added dropwise. The slurries were stirredat 40° C. for ca. 1 hour prior to cooling to 5° C. at a rate of 0.1°C./min and holding at 5° C. for approximately 18 hours. Solid materialswere filtered and dried under vacuum at approximately 40° C. forapproximately 21 hours. Isolated materials were slurried inmethanol:water (80:20) in a 1:16 (w/v) ratio. The slurries weretemperature cycled between 20° C. and 50° C. for ca. 48 hours at a rateof 0.1° C./min, with a hold of 4 hours at each temperature for 3 cycles.Solid materials were filtered and dried under vacuum at approximately40° C. for approximately 20 hours. Isolated material analyzed by XRPD tohave a unique crystalline form, designated to be Form 2.

Method VII. N-methylpyrrolidone:methanol:water (51:40:9) was added toBT-11 free base in a 53:1 (v/w) ratio to form a slurry by stirring for 1hour at 50° C. BT-11 dihydrochloride Form 2 was seeded into the mixture(approximately 4% weight). Approximately 2.2 equivalents of HCl wereadded dropwise. An additional approximate 4% weight of BT-11dihydrochloride Form 2 was added. The slurry was stirred at 50° C. forapproximately 18 hours prior to cooling to 5° C. at a rate of 0.1°C./min and holding at 5° C. for approximately 18 hours. Solid materialswere filtered. Isolated material analyzed by XRPD to have a uniquecrystalline form, designated to be Form 2.

Method VIII. N-methylpyrrolidone:methanol:water (51:40:9) or NMP:ethylacetate (81:19) were added to BT-11 free base in 53:1 and 63:1 (v/w)ratios, respectively, to form slurries by stirring for 1 hour at 50° C.BT-11 dihydrochloride Form 2 was seeded into the mixture (approximately4% weight). Approximately 2.2 equivalents of HCl were added dropwise. Anadditional approximate 4% weight of BT-11 dihydrochloride Form 2 wasadded. The slurry was stirred at 50° C. for approximately 18 hours priorto cooling to 5° C. at a rate of 0.1° C./min and holding at 5° C. forapproximately 18 hours. Solid materials were filtered. Isolatedmaterials were slurried in methanol:water (80:20) in approximately equalvolume to the original solvent system and stirred at 50° C. forapproximately 2 hours. The slurries were then cooled from 50° C. to 5°C. at a rate of 0.26° C./min. Solid materials were filtered and washedtwice with 10 volumes of methanol:water (80:20). Isolated material wasdried under vacuum at approximately 40° C. for approximately 64 hours.Isolated material analyzed by XRPD to have a unique crystalline form,designated to be Form 2.

Method IX. N-methylpyrrolidone:methanol (75:25) was added to BT-11 freebase in a 42:1 (v/w) ratio to form a slurry by stirring for 1 hour at50° C. BT-11 dihydrochloride Form 2 was seeded into the mixture(approximately 4% weight). Approximately 2.2 equivalents of HCl wereadded dropwise. An additional approximate 4% weight of BT-11dihydrochloride Form 2 was added. The slurry was stirred at 50° C. forapproximately 18 hours prior to cooling to 5° C. at a rate of 0.1°C./min and holding at 5° C. for approximately 18 hours. Solid materialswere filtered. Isolated materials were slurried in methanol:water(80:20) in approximately equal volume to the original solvent system andstirred at 50° C. for approximately 2 hours. The slurry was cooled from50° C. to 5° C. at a rate of 0.26° C./min. Solid materials were filteredand washed twice with 10 volumes of methanol:water (80:20). Isolatedmaterial was dried under vacuum at approximately 40° C. forapproximately 64 hours. Isolated material was slurried in methanol:water(80:20) in a 1:16 (w/v) ratio. The slurry was temperature cycled between20° C. and 50° C. for ca. 48 hours at a rate of 0.1° C./min with a holdof 4 hours at each temperature for 3 cycles. Solid material was filteredand analyzed by XRPD to have a unique crystalline form, designated to beForm 2.

Method X. N-methylpyrrolidone, NMP:water (90:10), NMP:methanol:water(51:40:9), or DMSO:methanol:water (50:40:10) were added to BT-11 freebase in 23:1, 20:1, 40:1, or 37:1 (v/w) ratios to form slurries bystirring for 1 hour at 55° C. BT-11 dihydrochloride Form 2 was seededinto the mixture (approximately 0% to 10% weight). Approximately 2.2equivalents of HCl were added dropwise. An additional approximate 0% to5% weight of BT-11 dihydrochloride Form 2 was added. The slurry wasstirred at 55° C. for approximately 5 hours prior to cooling to 5° C. ata rate of 0.1° C./min and holding at 5° C. for approximately 20 hours.Solid materials were filtered. Isolated materials from NMP were washedin NMP. Isolated materials from the other solvent systems were washedonce with methanol. Isolated material was dried under vacuum atapproximately 40° C. for approximately 38 hours and analyzed by XRPD tohave a unique crystalline form, designated to be Form 2.

Method XI N-methylpyrrolidone:water (90:10) was added to BT-11 free basein a 17:1 (v/w) ratio to form a slurry by stirring for 1 hour at 60° C.Approximately 2.2 equivalents of HCl were added dropwise in threealiquots. After each aliquot, 1% to 5% weight of BT-11 dihydrochlorideForm 2 was added as seed. The slurry was stirred at 60° C. forapproximately 21 hours prior to cooling to 5° C. at a rate of 0.25°C./min and holding at 5° C. for approximately 5 hours. Solid materialswere filtered. Isolated materials were washed once with methanol.Isolated material was dried under vacuum at approximately 40° C. forapproximately 64 hours and analyzed by XRPD to have a unique crystallineform, designated to be Form 2.

Method XII. DMSO:water (90:10) was added to BT-11 free base in a 17:1(v/w) ratio to form a slurry by stirring for 1 hour at 60° C.Approximately 2.2 equivalents of HCl were added dropwise in a singlealiquot. Approximately 6% weight of BT-11 dihydrochloride Form 2 wasadded as seed. The slurry was stirred at 60° C. for approximately 21hours prior to cooling to 5° C. at a rate of 0.25° C./min and holding at5° C. for approximately 5 hours. Solid materials were filtered. Isolatedmaterials were washed once with methanol. Isolated material was driedunder vacuum at approximately 40° C. for approximately 64 hours andanalyzed by XRPD to have a unique crystalline form, designated to beForm 2.

Method XIII. N-methylpyrrolidone:water (90:10) was added to BT-11 freebase in a 17:1 (v/w) ratio to form a slurry by stirring for 1 hour at60° C. Approximately 2.2 equivalents of HCl were added dropwise in threealiquots. After each aliquot, 0.4% to 1% weight of BT-11 dihydrochlorideForm 2 was added as seed. The slurry was stirred at 60° C. forapproximately 21 hours prior to cooling to 5° C. at a rate of 0.25°C./min and holding at 5° C. for approximately 4 hours. Solid materialswere filtered. Isolated materials were washed twice with methanol.Isolated material was dried under vacuum at approximately 40° C. forapproximately 64 hours and analyzed by XRPD to have a unique crystallineform, designated to be Form 2.

Method XIV. DMSO:water (90:10) was added to BT-11 free base in a 34:1(v/w) ratio to form a slurry by stirring for 1 hour at 60° C.Approximately 2.2 equivalents of HCl were added dropwise in a singlealiquot. Approximately 3.4% weight of BT-11 dihydrochloride Form 2 wasadded as seed in two aliquots. The slurry was stirred at 60° C. forapproximately 21 hours prior to cooling to 5° C. at a rate of 0.25°C./min and holding at 5° C. for approximately 5 hours. Solid materialswere filtered. Isolated materials were washed twice with methanol.Isolated material was dried under vacuum at approximately 40° C. forapproximately 64 hours and analyzed by XRPD to have a unique crystallineform, designated to be Form 2.

Method XV. NMP:water (90:10) was added to BT-11 free base in a 15:1(v/w) ratio to form a slurry by stirring at 65° C. for approximately 45minutes. Slurry was cooled to 60° C. over approximately 5 minutes.Approximately 1.1 equivalents of HCl was added to the slurry at a rateof 0.9 volumes/hour. 2% weight of BT-11 dihydrochloride Form 2 wasadded. After 1 hour, approximately 1.1 equivalents of HCl was added in 4aliquots at a rate of 0.46 volumes/hour, pausing the acid additionbetween each aliquot for approximately 20 minutes. The slurry wasstirred at 60° C. for approximately 18 hours. The experiment was cooledto 5° C. at a rate of 0.1° C./min and held at 5° C. for 20 hours. Solidmaterial was filtered and washed with 2 volumes of methanol. Wet solidwas dried under vacuum at 40° C. for approximately 82 hours. Isolatedmaterial was analyzed by XRPD to have a unique crystalline form,designated to be Form 2.

Method XVI. DMSO:methanol:water (50:40:10) was added to BT-11 free basein a 34:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 45 minutes. Approximately 2.2 equivalents of HCl was addedto the slurry at a rate of 4.6 volumes/hour. Slurry was cooled to 60° C.over approximately 5 minutes. 2% weight of BT-11 dihydrochloride Form 2was added. The slurry was stirred at 60° C. for approximately 18 hours.The experiment was cooled to 5° C. at a rate of 0.1° C./min and held at5° C. for 18 hours. Solid material was filtered and washed with 2volumes of methanol. Wet solid was dried under vacuum at 40° C. forapproximately 82 hours. Isolated material was analyzed by XRPD to have aunique crystalline form, designated to be Form 2.

Method XVII. DMSO:methanol:water (50:40:10) was added to BT-11 free basein a 40:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 1 hour. Approximately 2.63 equivalents of HCl was added tothe slurry dropwise over 20 minutes. Slurry was cooled to 60° C. overapproximately 5 minutes. 2% weight of BT-11 dihydrochloride Form 2 wasadded. The slurry was stirred at 60° C. for approximately 8 hours. Theexperiment was cooled to 20° C. at a rate of 0.1° C./min and held at 20°C. for 10 hours. The slurry was heated to 50° C. at a rate of 0.5°C./min and held at 50° C. for 4 hours. The slurry was cooled to 5° C. at0.1° C./min and held at 5° C. for 3 hours. Solid material was filteredand washed with 2 volumes of methanol. Wet solid was dried under vacuumat 40° C. for approximately 82 hours. Isolated material was analyzed byXRPD to have a unique crystalline form, designated to be Form 2.

Method XVIII. DMSO:methanol:water (50:40:10) was added to BT-11 freebase in a 40:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 1 hour. Approximately 2.63 equivalents of HCl was added tothe slurry dropwise over 20 minutes. Slurry was cooled to 60° C. overapproximately 5 minutes. 2% weight of BT-11 dihydrochloride Form 2 wasadded. The slurry was stirred at 60° C. for approximately 3 hours. Waterwas added at a rate of 2 volumes/hour at 60° C. to reach a solventsystem of 34:27:39 and the slurry was stirred for an additional 1.5hours. The slurry was cooled to 5° C. at 0.1° C./min and held at 5° C.for 8 hours. Solid material was filtered and washed with 2 volumes ofmethanol. Wet solid was dried under vacuum at 40° C. for approximately86 hours. Isolated material was analyzed by XRPD to have a uniquecrystalline form, designated to be Form 2.

Method XIX. DMSO:methanol:water (50:40:10) was added to BT-11 free basein a 40:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 45 minutes. Approximately 2.63 equivalents of HCl wasadded to the slurry at a rate of 2 volumes/hour. Slurry was stirred at65° C. for approximately 45 minutes. A hot polish filtration wasconducted. The filtrate was heated to 60° C. 2% weight of BT-11dihydrochloride Form 2 was added. The slurry was stirred at 60° C. forapproximately 7 hours. The slurry was cooled to 5° C. at 0.1° C./min andheld at 5° C. for 9 hours. The slurry was cooled to 0° C. at a rate of0.1° C./min and stirred at 0° C. for 16 hours. Solid material wasfiltered and washed with 1 volume of methanol. Wet solid was dried undervacuum at 40° C. for approximately 96 hours. Isolated material wasanalyzed by XRPD to have a unique crystalline form, designated to beForm 2.

Method XX DMSO:methanol:water (50:40:10) was added to BT-11 free base ina 33:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 45 minutes. Approximately 2.1 equivalents of HCl was addedto the slurry at a rate of 2 volumes/hour. Slurry was stirred at 65° C.for approximately 30 minutes. A hot polish filtration was conducted. Thefiltrate was heated to 60° C. 1% weight of BT-11 dihydrochloride Form 2was added. The slurry was stirred at 60° C. for approximately 8 hours.The slurry was cooled to 0° C. at a rate of 0.1° C./min and stirred at0° C. for 25 hours. Solid material was filtered and washed with 1 volumeof methanol. Wet solid was dried under vacuum at 40° C. forapproximately 90 hours. Isolated material was analyzed by XRPD to have aunique crystalline form, designated to be Form 2.

Method XXI. DMSO:methanol:water (50:40:10) was added to BT-11 free basein a 33:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 1 hour. Approximately 2.6 equivalents of HCl was added tothe slurry at a rate of 2 volumes/hour. The slurry was cooled to 60° C.1% weight of BT-11 dihydrochloride Form 2 was added. The slurry wasstirred at 60° C. for approximately 8 hours. The slurry was cooled to 0°C. at a rate of 0.1° C./min and stirred at 0° C. for 4 hours. Solidmaterial was filtered and washed with 1 volume of methanol. Wet solidwas dried under vacuum at 40° C. for approximately 68 hours. Isolatedmaterial was analyzed by XRPD to have a unique crystalline form,designated to be Form 2.

Method XXII. DMSO:methanol:water (50:40:10) was added to BT-11 free basein a 33:1 (v/w) ratio to form a slurry by stirring at 65° C. forapproximately 1 hour. Approximately 2.2 equivalents of HCl was added tothe slurry at a rate of 2 volumes/hour. The slurry was cooled to 60° C.1% weight of BT-11 dihydrochloride Form 2 was added. The slurry wasstirred at 60° C. for approximately 8 hours. The slurry was cooled to 0°C. at a rate of 0.1° C./min and stirred at 0° C. for 24 hours. Solidmaterial was filtered and washed with 1 volume of methanol. Wet solidwas dried under vacuum at 40° C. for approximately 68 hours. Isolatedmaterial was analyzed by XRPD to have a unique crystalline form,designated to be Form 2.

Method XXIII. BT-11 free base was added to DMSO:methanol:water(50:40:10) in a 1:33 (w/v) ratio to form a slurry by stirring at 25-35°C. for approximately 10 minutes. The temperature of the reaction masswas raised to 60-65° C. Approximately 2 to 3 equivalents of HCl,prepared in DMSO:methanol:water (50:40:10), were added over 1.5 to 2.5hours. The reaction mass was filtered at 60-65° C. Approximately 1 to 2percent weight of BT-11 dihydrochloride Form 2 was added. The slurry wasstirred at 60-65° C. for approximately 6 to 8 hours. The slurry wascooled to 20-30° C. at a rate of approximately 8° C./hour and stirred at20 to 30° C. for 1 to 2 hours. The slurry was cooled to 0 to 5° C. at arate of approximately 6° C./hour and stirred at 0 to 5° C. for 12 to 14hours. Solid material was filtered. Wet material was slurried inmethanol:water for 5 to 96 hours at 25 to 55° C. Wet solid was filteredand dried under vacuum at 25 to 55° C. Isolated material was analyzed byXRPD to have a unique crystalline form, designated to be Form 2.

Characterization

BT-11 Form 2 was characterized via XRPD (FIGS. 11, 12, 13, and 14).Methods I, II, and III are captured in FIG. 11. Methods IV and V arecaptured by FIG. 12. Method VIII is captured in FIG. 13. Methods XV,XVI, XVII, XVIII, XIX, XX, XXI and XXII are captured in FIGS. 14A-14B.No major differences in pattern were identified for methods VI, VII, IX,X, XI, XII, XIII or XIV. Representative diffraction peaks are presentedin Table 5. Representative reduced cell parameters are presented inTable 6.

TABLE 5 Diffraction peaks observed for Form 2 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 7.1488 12.36575 623.69 47.22 24.84 1 0 07.1443 2 10.0477 8.80367 774.17 68.38 30.83 1 1 0 10.0306 3 11.96217.39867 489.53 37.06 19.49 0 1 −1 11.9588 4 12.564 7.04553 952.4 84.1337.93 1 1 −1 12.5639 5 14.322 6.18441 723.14 54.75 28.8 2 0 0 14.3165 615.2001 5.82907 332.76 25.19 13.25 1 1 1 15.201 7 16.607 5.33829 673.3750.98 26.81 2 1 −1 16.6079 8 17.1144 5.18115 966.5 97.57 38.49 0 2 −117.1117 9 19.3787 4.58057 689.28 60.88 27.45 0 0 2 19.3915 10 20.66094.2991 1607.15 162.24 64 2 2 −1 20.6553 11 21.5528 4.12316 278.52 21.0911.09 3 0 0 21.5457 12 22.3362 3.977 1114.59 139.1 44.38 3 1 −1 22.333913 23.3545 3.80586 2511.24 417.87 100 0 3 1 23.3463 14 23.9761 3.70857466.24 48.49 18.57 2 2 1 23.9873 15 25.5263 3.48675 656.09 122.82 26.133 2 −1 25.5218 16 28.4062 3.13947 2488.5 414.09 99.09 0 4 0 28.3986 1728.863 3.09081 332.59 96.85 13.24 4 0 0 28.8628 18 29.3245 3.04321568.51 70.95 22.64 1 4 0 29.3203 19 29.8776 2.98812 391.79 48.89 15.6 21 −3 29.8983 20 34.6459 2.58701 450.07 56.17 17.92 2 3 2 34.6701

TABLE 6 Reduced Cell Parameters of Form 2 of BT-11 dihydrochloride. CellParameters Value a sigma [Å] 12.81 b sigma [Å] 12.56 c sigma [Å] 9.48alpha (sigma) [°] 90 beta (sigma) [°] 105.25 gamma (sigma) [°] 90

BT-11 Form 2 was characterized via thermal gravimetric trace (FIG. 15).The thermal gravimetric trace (TG) showed no loss in mass below 100° C.Consecutive mass losses of 5.7 and 5.9% were observed. Two broadendothermic events associated with these losses were noted with onsetsof ˜128 and 211° C., respectively. An exothermic event was observedpost-salt disproportionation with onset ˜257° C. Post-recrystallization,an endothermic event with onset ˜315° C. was noted. A representativeTG/DTA for Methods XV and XIX is depicted in FIG. 16 and arepresentative TG/DTA for Methods XVI, XVII, XVIII, XX, XXI and XXII isdepicted in FIG. 17. In FIG. 17, the material showed consecutive masslosses from the outset to ca. 35° C. An initial mass loss of 0.1% wasobserved, with further mass losses of 5.4% and 5.2% between 140-250° C.The larger mass losses were associated with broad endothermic events,with degradation of the material observed above 250° C.

BT-11 Form 2 was characterized via PLM. The PLM images showed someblock-like particles with aggregation visible. The material appearedbirefringent under polarized light, indicative of a crystallinematerial. PLM analysis of the dried material showed birefringentmaterial with a mixture of small and large, plate-like morphology, withsome aggregation observed.

BT-11 Form 2 was characterized via DSC (FIG. 18). The DSC thermogramshowed a broad endothermic event (onset at 145° C.) observed which wasimmediately followed by a second broad endothermic event (onset at 226°C.). An exothermic event post-disproportionation was observed (onset at248° C.). There were 2 sharp endothermic events were observed (onsets at315 and 328° C., respectively).

BT-11 Form 2 was characterized via DVS (FIGS. 19A-19B). DVS analysisshowed a total 0.32% mass increase up to 90% RH. There was a 0.23% massuptake on 2nd cycle and the material was slightly hygroscopic (FIG.19A). Analysis of the DVS kinetic plot showed no evidence ofre-crystallization or form change occurring during the DVS experiment(FIG. 19B).

BT-11 Form 2 was characterized by PSD. PSD analysis (FIG. 20) of thedried material showed that the material had a d₉₀ of 83.9 μm, d₅₀ of31.9 μm and d₁₀ of 18.2 μm.

Example 4. Crystal Form 3 of BT-11 Form 3 Preparation Methods

Methanol, 1,4-dioxane, 1-butanol, 1-propanol, 2-methyl tetrahydrofuran,butyl acetate, dichloromethane, ethyl acetate, isopropyl alcohol, methylethyl ketone, tert-butyl methyl ether, or tetrahydrofuran wereindependently added to BT-11 dihydrochloride in a 100:1 (v/w) ratio toform slurries. Slurries were thermally cycled with agitation for 72hours between ambient temperature for 4 hours followed by 40° C. for 4hours. Solid material was isolated by filtration and dried at 40° C. for2 hours. Dried material was analyzed by XRPD to have a uniquecrystalline form, designated to be Form 3.

Alternatively, lyophilized BT-11 dihydrochloride was added to ethylacetate in a 1:50 (w/v) ratio to form a slurry. Slurry was thermallycycled with agitation for 24 hours between ambient temperature for 4hours followed by 40° C. for 4 hours. Solid material was isolated byfiltration and dried at ambient temperature under vacuum for 3 hours.

Characterization

BT-11 Form 3 was characterized via XRPD (FIGS. 21A, 21B, 21C, and 21D).Crystallinity varied slightly between samples. Amorphous forms wereobserved from 1,4-dioxane, 1-butanol, 1-propanol, isopropyl alcohol,tert-butyl methyl ether and tetrahydrofuran solvents prior to drying.Minor additional peaks were noted in patterns produced from methanol,ethyl acetate, isopropyl acetate and MEK. Replacement of the listedsolvents with acetone or chloroform resulted in mixtures of Forms 2 and3 and Forms 1 and 3, respectively. Diffraction peaks for Form 3 arepresented in Table 7. Reduced cell parameters for Form 3 are presentedin Table 8.

TABLE 7 Diffraction peaks observed for Form 3 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 8.1744 10.81637 465.03 93.89 46.13 0 1 18.1694 2 10.263 8.61943 1008.11 127.21 100 0 0 1 10.2418 3 11.45947.72206 351.81 39.95 34.9 0 1 0 11.4459 4 14.0108 6.32108 526.47 93.0152.22 1 2 0 13.9276 5 14.5785 6.07617 186.6 37.67 18.51 0 1 2 14.5966 615.3272 5.78102 314.07 71.34 31.15 1 1 −2 15.3002 7 17.1351 5.17494746.56 113.05 74.06 0 2 1 17.0862 8 17.9941 4.92979 144.46 36.46 14.33 21 −2 18.0093 9 20.5635 4.31924 298.21 60.21 29.58 0 0 2 20.5666 1021.387 4.15475 149.78 45.36 14.86 0 2 3 21.3951 11 22.4595 3.95873333.25 67.28 33.06 1 3 0 22.4057 12 23.036 3.86094 444.07 56.04 44.05 02 0 23.008 13 23.9527 3.71522 569.78 86.28 56.52 2 1 −4 23.9508 1424.4536 3.64025 773.76 273.39 76.75 1 1 −3 24.4152 15 25.0495 3.55498603.51 152.31 59.87 2 −1 −4 25.0544 16 27.9172 3.19598 845 191.93 83.821 3 −1 28.0041 17 29.0479 3.0741 687.16 138.74 68.16 0 3 4 29.0549 1830.4717 2.9312 354.82 42.58 35.2 1 1 2 30.4797 19 31.4541 2.84421 405.58163.77 40.23 0 2 −1 31.4419 20 34.1203 2.62782 476.15 72.1 47.23 0 1 434.1199

TABLE 8 Reduced Cell Parameters of Form 3 of BT-11 dihydrochloride. CellParameters Value a sigma [Å] 9.30 b sigma [Å] 11.78 c sigma [Å] 10.20alpha (sigma) [°] 71.15 beta (sigma) [°] 106.99 gamma (sigma) [°] 108.35

BT-11 Form 3 was characterized via thermal gravimetric trace (FIG. 22).The thermal gravimetric trace (TG) showed a 5.6% loss in mass below 100°C. A broad endothermic event (onset ˜61° C.) was associated with thisstep. A mass loss of ˜10.7% was observed around 140° C. to just above260° C. A broad endothermic event was observed (onset ˜230° C.). A sharpendothermic event (onset ˜312° C.) was observed.

In PLM, the PLM images showed that the material consisted of no clearmorphology and aggregation was visible. The material appearedbirefringent under polarized light, indicative of a crystallinematerial.

BT-11 Form 3 was characterized via differential scanning calorimetry(FIG. 23). The DSC thermogram showed a broad endothermic event (onset at99° C.). A second broad endothermic event was also noted (onset ˜261°C.). Two sharp endothermic events were observed post-disproportionation(onsets at 318 and 328° C., respectively).

BT-11 Form 3 was characterized via dynamic vapor sorption. (FIGS.24A-24B). DVS analysis showed a total 7.8% mass increase up to 90% RH(FIG. 24A). Only 2.8% of total moisture uptake was due to surfacemoisture. 5.0% of total mass was lost below 20% RH. Rehydration appearedslower than dehydration. No evidence of re-crystallization or formchange occurring during the DVS experiment (FIG. 24B). Dehydration stepwas more pronounced that the rehydration step.

Example 5. Crystal Form 4 of BT-11 Form 4 Preparation Methods

Acetic acid was added to BT-11 dihydrochloride in a 100:1 (v/w) ratio toform a slurry. Slurry was thermally cycled with agitation for 72 hoursbetween ambient temperature for 4 hours followed by 40° C. for 4 hours.Solid material was isolated by filtration and analyzed by XRPD to have aunique crystalline form, designated to be Form 4.

Characterization

BT-11 Form 4 was characterized via XRPD (FIG. 25). Diffraction peaks arepresented in Table 9.

TABLE 9 Diffraction peaks observed for Form 4 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. No. [°2θ] [Å] [cts] [cts *°2θ] [%]1 3.5565 24.84392 203.22 15.39 22.06 2 7.1292 12.39974 573.9 57.94 62.33 8.6264 10.25064 242.81 21.45 26.36 4 13.1455 6.73515 696.7 96.71 75.635 14.2953 6.1959 302.47 34.35 32.83 6 17.8608 4.96626 110.89 27.99 12.047 19.0407 4.66111 162.23 16.38 17.61 8 20.3714 4.35955 301.82 38.0932.76 9 20.6541 4.30051 248.6 37.64 26.99 10 21.0797 4.21462 119.7 18.1312.99 11 21.9166 4.05554 186.13 32.88 20.2 12 22.4612 3.95843 249.1950.31 27.05 13 23.6032 3.76944 921.22 302.24 100 14 24.5428 3.62721186.76 47.13 20.27 15 25.4064 3.50584 243.11 73.63 26.39 16 26.03083.42314 149.89 45.39 16.27 17 26.8957 3.31225 87.21 10.47 9.47 1827.4974 3.24381 422.07 37.28 45.82 19 29.9564 2.9829 93.81 14.21 10.1820 30.4171 2.93877 80.15 12.14 8.7

Example 6. Crystal Form 5 of BT-11 Form 5 Preparation Methods

Acetonitrile was added to BT-11 dihydrochloride in a 100:1 (v/w) ratioto form a slurry. Slurry was thermally cycled with agitation for 72hours between ambient temperature for 4 hours followed by 40° C. for 4hours. Solid material was isolated by filtration and analyzed by XRPD tohave a unique crystalline form, designated to be Form 5.

Characterization

BT-11 Form 5 was characterized via XRPD (FIG. 26). Diffraction peaks arepresented in Table 10.

TABLE 10 Diffraction peaks observed for Form 5 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. No. [°2θ] [Å] [cts] [cts *°2θ] [%]1 3.6941 23.91843 1530.44 96.56 64.49 2 7.0177 12.59648 460.23 58.0719.39 3 7.3929 11.958 2373.27 179.69 100 4 11.0881 7.97978 447.47 28.2318.85 5 13.4866 6.56555 699.4 70.6 29.47 6 13.9403 6.3529 536.93 60.9822.62 7 14.8072 5.98285 1224.35 154.5 51.59 8 19.8257 4.47828 319.424.18 13.46 9 20.1935 4.39754 482.22 48.68 20.32 10 20.736 4.28371464.46 58.61 19.57 11 21.3206 4.16756 574.55 87 24.21 12 21.8298 4.07147528.2 33.33 22.26 13 22.5514 3.9428 582.66 102.93 24.55 14 22.87973.88696 681.69 103.22 28.72 15 23.5297 3.78104 720.97 163.76 30.38 1624.7009 3.60435 967.04 195.24 40.75 17 25.3182 3.51786 256.23 25.87 10.818 27.1604 3.28329 731.5 92.31 30.82 19 27.422 3.25256 643.43 97.4327.11 20 28.2596 3.15803 439.56 77.65 18.52

BT-11 Form 5 was characterized via thermal gravimetric trace (FIG. 27).The thermal gravimetric trace (TG) showed a 5.0% loss in mass below 150°C. A mass loss of ˜11.3% was observed above 150° C. to just above 250°C. A broad endothermic event was observed (onset ˜202° C.). A sharpendothermic event (onset ˜328° C.) was observed.

Example 7. Crystal Form 6 of BT-11 Form 6 Preparation Methods

Water was added to BT-11 dihydrochloride in a 100:1 (v/w) ratio to forma slurry. Slurry was thermally cycled with agitation for 72 hoursbetween ambient temperature for 4 hours followed by 40° C. for 4 hours.Solid material was isolated by filtration and analyzed by XRPD to have aunique crystalline form, designated to be Form 6.

Characterization

BT-11 Form 6 was characterized via XRPD (FIG. 28). Diffraction peaks arepresented in Table 11. Reduced cell parameters are presented in Table12.

TABLE 11 Diffraction peaks observed for Form 6 of BT-11. Pos. d-spacingHeight Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts] [cts *°2θ] [%]h k l [°2θ] 1 3.2893 26.86163 129.02 104.2 6.92 0 0 1 3.8177 2 7.494311.79648 68.65 10.4 3.68 0 0 2 7.6396 3 9.4388 9.37014 772.3 48.73 41.420 1 1 9.4362 4 9.8112 9.01531 358.81 45.28 19.24 0 1 0 9.7881 5 13.95876.34457 356.46 35.98 19.12 1 0 1 13.7132 6 14.9672 5.91927 303.73 107.3216.29 1 0 2 15.2391 7 15.6759 5.65318 206.9 41.77 11.1 0 0 4 15.3135 816.3126 5.42946 145.55 17.47 7.81 0 1 2 16.3131 9 16.9298 5.23722 294.1389.08 15.77 0 1 −1 16.9293 10 17.5467 5.05026 71.65 8.6 3.84 1 0 317.4952 11 18.9455 4.68431 379.61 47.9 20.36 0 0 5 19.1743 12 19.64534.51899 128.28 32.37 6.88 0 2 0 19.6486 13 20.4165 4.35 96.87 12.22 5.191 0 4 20.2471 14 21.2313 4.18487 109.82 55.43 5.89 1 0 0 21.2313 1524.3455 3.65616 68.78 34.71 3.69 1 1 2 24.1799 16 25.737 3.46155 155.4647.08 8.34 1 1 3 25.6879 17 26.7952 3.3272 1864.76 705.92 100 2 0 126.7938 18 27.7537 3.21443 1319.22 332.94 70.74 1 1 4 27.6702 19 28.27763.15606 481.78 97.27 25.84 1 2 0 28.2776 20 29.1773 3.06076 126.16 31.846.77 2 0 3 28.9689

TABLE 12 Reduced Cell Parameters of Form 6 of BT-11 (suspected freebase). Cell Parameters Value a sigma [Å] 4.21 b sigma [Å] 10.65 c sigma[Å] 10.47 alpha (sigma) [°] 117.79 beta (sigma) [°] 89.76 gamma (sigma)[°] 84.35

BT-11 Form 6 was characterized via thermal gravimetric trace (FIG. 29).The thermal gravimetric trace (TG) showed a 15.4% loss in mass belowaround 130° C. A mass loss of 1.3% was observed above 150° C. to justbelow 250° C. with a weak exothermic event noted during the loss in mass(onset ˜180° C.). A sharp endothermic event (onset ˜328° C.) wasobserved.

Example 8. Crystal Form 7 of BT-11 Form 7 Preparation Methods

Methanol:water (80:20) was added to BT-11 dihydrochloride in a 40:1(v/w) ratio to form a slurry. Slurry was thermally cycled with agitationfor 72 hours between ambient temperature for 4 hours followed by 40° C.for 4 hours. Solid material was isolated by filtration and analyzed byXRPD to have a unique crystalline form, designated to be Form 7.

Characterization

BT-11 Form 7 was characterized via XRPD (FIG. 30). Post-drying, BT-11Form 7 was observed to convert to Form 2. Diffraction peaks arepresented in Table 13. Reduced cell parameters are presented in Table14.

TABLE 13 Diffraction peaks observed for Form 7 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 9.4586 9.35057 152.63 15.41 18.88 0 1 09.4629 2 10.0617 8.79144 557.22 28.13 68.94 1 0 0 10.0481 3 12.26147.2187 723.5 36.52 89.51 0 1 1 12.2943 4 13.6865 6.4701 168.28 16.9920.82 0 1 −1 13.6472 5 14.2039 6.23557 200.82 15.2 24.84 1 1 1 14.729 615.1273 5.85695 148.7 22.52 18.4 1 1 0 15.121 7 18.5131 4.79272 177.117.88 21.91 2 0 1 18.4052 8 20.2243 4.39092 172.42 15.23 21.33 2 0 020.1745 9 20.6669 4.29786 346.38 30.6 42.85 1 −2 1 20.7074 10 22.50783.95033 224.87 28.38 27.82 1 −2 −1 22.5086 11 22.7248 3.91311 185.7914.07 22.98 1 2 1 22.5882 12 24.5678 3.62358 191.1 24.11 23.64 1 0 324.4743 13 24.9061 3.57512 298.63 15.07 36.94 2 −2 0 24.9162 14 25.93583.43547 392.06 34.63 48.5 1 1 3 25.9976 15 27.1472 3.28486 808.31 91.8100 1 1 −2 27.1268 16 27.5788 3.23442 208.04 21 25.74 0 1 3 27.5554 1731.5353 2.83707 158.95 20.06 19.66 1 3 1 31.5456 18 31.5353 2.83707158.95 20.06 19.66 2 −1 3 31.4949 19 32.6526 2.7425 138.8 14.01 17.17 3−2 0 32.6918 20 32.6526 2.7425 138.8 14.01 17.17 0 1 −4 32.6523

TABLE 14 Reduced Cell Parameters of Form 7 of BT-11 dihydrochloride.Cell Parameters Value a sigma [Å] 9.78 b sigma [Å] 9.53 c sigma [Å]10.91 alpha (sigma) [°] 88.50 beta (sigma) [°] 66.51 gamma (sigma) [°]99.77

Example 9. Crystal Form 8 of BT-11 Form 8 Preparation Methods

2-propanol was added to BT-11 dihydrochloride in a 100:1 (v/w) ratio toform a slurry. Slurry was thermally cycled with agitation for 72 hoursbetween ambient temperature for 4 hours followed by 40° C. for 4 hours.Solid material was isolated by filtration and dried at 40° C. for 2hours. Dried material was analyzed by XRPD to have a unique crystallineform, designated to be Form 8.

Characterization

BT-11 Form 8 was characterized via XRPD (FIG. 31). Diffraction peaks arepresented in Table 15.

TABLE 15 Diffraction peaks observed for Form 8 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. No. [°2θ] [Å] [cts] [cts *°2θ] [%]1 6.6209 13.35038 315.65 19.92 29.09 2 7.5707 11.6776 1085.17 82.16 1003 11.3551 7.79276 227.61 8.62 20.97 4 13.2915 6.66149 614.77 38.79 56.655 13.5731 6.52393 586.72 44.42 54.07 6 15.1814 5.83621 540.27 61.3649.79 7 19.5347 4.54434 450.02 34.07 41.47 8 22.2588 3.99396 847.58149.73 78.11 9 22.933 3.87805 444.11 67.25 40.93 10 23.5098 3.7842323.89 40.87 29.85 11 24.0544 3.69974 710.31 107.56 65.46 12 24.48863.63512 320.6 48.55 29.54 13 25.3358 3.51545 555.82 112.22 51.22 1425.8734 3.44361 258.38 39.13 23.81 15 26.6435 3.3458 322.06 56.9 29.6816 27.451 3.24919 424.16 85.64 39.09 17 27.9198 3.19569 524.57 66.1948.34 18 29.1013 3.06858 300.27 53.05 27.67 19 30.3273 2.94727 275.8248.73 25.42 20 32.3621 2.76645 226.25 34.26 20.85

BT-11 Form 8 was characterized via thermal gravimetric trace (FIG. 32).The thermal gravimetric trace showed consecutive losses in mass observedfrom the outset of heating up to −250° C. The 1.7% and 3.0% mass losseswere observed. A 9.0% mass loss was observed. The differentialthermogram (TG) showed a broad exothermic event (onset 230° C.) followedby two endothermic events (onsets of 317 and 329° C.). No further lossin mass until the onset of the first endothermic event observed.

Example 10. Crystal Form 9 of BT-11 Form 9 Preparation Methods

Ethanol was added to BT-11 dihydrochloride in a 100:1 (v/w) ratio toform a slurry. Slurry was thermally cycled with agitation for 72 hoursbetween ambient temperature for 4 hours followed by 40° C. for 4 hours.Solid material was isolated by filtration and dried at 40° C. for 2hours. Dried material was analyzed by XRPD to have a unique crystallineform, designated to be Form 9.

Characterization

BT-11 Form 9 was characterized via XRPD (FIG. 33). Diffraction peaks arepresented in Table 16. Reduced cell parameters are presented in Table17.

TABLE 16 Diffraction peaks observed for Form 9 of BT-11 dihydrochloride.Pos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 9.4586 9.35057 152.63 15.41 18.88 0 1 09.4629 2 10.0617 8.79144 557.22 28.13 68.94 1 0 0 10.0481 3 12.26147.2187 723.5 36.52 89.51 0 1 1 12.2943 4 13.6865 6.4701 168.28 16.9920.82 0 1 −1 13.6472 5 14.2039 6.23557 200.82 15.2 24.84 1 1 1 14.729 615.1273 5.85695 148.7 22.52 18.4 1 1 0 15.121 7 18.5131 4.79272 177.117.88 21.91 2 0 1 18.4052 8 20.2243 4.39092 172.42 15.23 21.33 2 0 020.1745 9 20.6669 4.29786 346.38 30.6 42.85 1 −2 1 20.7074 10 22.50783.95033 224.87 28.38 27.82 1 −2 −1 22.5086 11 22.7248 3.91311 185.7914.07 22.98 1 2 1 22.5882 12 24.5678 3.62358 191.1 24.11 23.64 1 0 324.4743 13 24.9061 3.57512 298.63 15.07 36.94 2 −2 0 24.9162 14 25.93583.43547 392.06 34.63 48.5 1 1 3 25.9976 15 26.0429 3.42159 1698.52 192.948.45 4 −3 −3 26.0557 16 27.1381 3.28594 1849.07 209.99 52.74 1 −5 −227.1918 17 27.1472 3.28486 808.31 91.8 100 1 1 −2 27.1268 18 27.57883.23442 208.04 21 25.74 0 1 3 27.5554 19 27.64 3.2274 1658.27 188.3347.3 5 −2 0 27.6262 20 31.5353 2.83707 158.95 20.06 19.66 1 3 1 31.5456

TABLE 17 Reduced Cell Parameters of Form 9 of BT-11 dihydrochloride.Cell Parameters Value a sigma [Å] 16.93 b sigma [Å] 17.31 c sigma [Å]10.08 alpha (sigma) [°] 100.23 beta (sigma) [°] 100.89 gamma (sigma) [°]71.77

BT-11 Form 9 was characterized via thermal gravimetric trace (FIG. 34).The thermal gravimetric trace (TG) showed a 9.6% loss in mass belowaround 90° C. Consecutive mass losses of 4.0 and 5.1% were observed. Abroad endothermic event (onset ˜306° C.) followed by a sharp endothermicevent (onset ˜330°) was observed.

Example 11. Crystal Form 10 of BT-11 Form 10 Preparation Methods

Form 3 was mixed with pH 1.2 buffer and shaken at 37° C. for 24 hours.pH 1.2 buffer was prepared mixing 0.2 M hydrochloric acid, 0.2 Mpotassium chloride and water in an 18:6:1 ratio. Remaining solids wereisolated by filtration and analyzed by XRPD to have a unique crystallineform, designated to be Form 10.

Characterization

BT-11 Form 10 was characterized via XRPD (FIG. 35). Diffraction peaksare presented in Table 18. Reduced cell parameters are presented inTable 19.

TABLE 18 Diffraction peaks observed for Form 10 of BT-11 dihydrochloridePos. d-spacing Height Area Rel. Int. Position Calc. No. [°2θ] [Å] [cts][cts *°2θ] [%] h k l [°2θ] 1 8.9826 9.84494 684.44 77.73 100 1 2 08.9665 2 9.6234 9.19079 649.47 73.76 94.89 0 0 1 9.635 3 10.4558 8.46094233.56 41.26 34.12 1 1 −1 10.4658 4 12.9085 6.85826 154.48 19.49 22.57 22 0 12.9046 5 13.7375 6.44621 151.94 30.68 22.2 1 2 1 14.0958 6 14.51126.1042 238.72 60.25 34.88 0 3 −1 14.4936 7 16.9278 5.23784 150.48 15.1921.99 3 1 −1 17.0269 8 17.5253 5.06059 313.71 47.5 45.83 1 4 −1 17.47799 19.3197 4.59441 359.52 54.44 52.53 0 0 2 19.339 10 21.6188 4.1107495.48 14.46 13.95 1 1 2 21.6128 11 23.2332 3.82862 358.23 54.24 52.34 32 −2 23.1768 12 23.9357 3.71781 221.79 33.58 32.41 1 3 2 23.9437 1325.4626 3.49824 299.3 60.43 43.73 2 2 2 25.4202 14 26.1681 3.40549 647.9130.81 94.66 1 5 −2 26.1582 15 27.5223 3.24094 569.78 100.66 83.25 4 3−2 27.5509 16 28.2002 3.16455 302.24 45.77 44.16 1 7 1 28.2007 1729.1394 3.06211 195.24 23.43 28.53 5 3 0 29.1151 18 29.7769 3.00048206.66 41.72 30.19 3 0 −3 29.774 19 31.5542 2 83542 231.62 46.76 33.84 60 −1 31.5527 20 33.2878 2.69161 85.5 25.89 12.49 4 2 2 33.2901

TABLE 19 Reduced Cell Parameters of Form 10 of BT-11 dihydrochloride.Cell Parameters Value a sigma [Å] 17.03 b sigma [Å] 24.55 c sigma [Å]9.45 alpha (sigma) [°] 90.00 beta (sigma) [°] 103.98 gamma (sigma) [°]90.00

Example 12. Crystal Form 11 of BT-11 Form 11 Preparation Methods

N-methylpyrrolidone or 90:10 (v/v) mixtures of NMP:ethanol,NMP:2-propanol, NMP:1-propanol, NMP:acetonitrile, NMP:acetone, NMP:ethylacetate, or NMP:water were added to BT-11 free base in 24:1, 36:1, 34:1,34:1, 45:1, 36:1, 36:1, or 26:1 (w/v) ratios, respectively, to formslurries by stirring for 1 hour at 50° C. The temperature was decreasedto 40° C., over approximately 10 minutes. 2.2 equivalents of HCl wereadded dropwise. The slurries were stirred at 40° C. for approximately 90minutes. The slurries were cooled to 5° C. at a rate of 0.1° C./min andtemperature cycled between 5° C. and 40° C. for 1 cycle (0.1° C./min)prior to holding at 5° C. for approximately 6 hours. Solid materialswere filtered and dried under vacuum at approximately 40° C. forapproximately 21 hours. Isolated material analyzed by XRPD to have aunique crystalline form, designated to be Form 11.

Characterization

BT-11 Form 11 was characterized via XRPD (FIG. 36).

Example 13. Crystal Form 12 of BT-11 Form 12 Preparation Methods

A 90:10 (v/v) mixture of NMP:methanol was added to BT-11 free base in32:1 to form a slurry by stirring for 1 hour at 50° C. The temperaturewas decreased to 40° C., over approximately 10 minutes. 2.2 equivalentsof HCl were added dropwise. The slurries were stirred at 40° C. forapproximately 90 minutes. The slurries were cooled to 5° C. at a rate of0.1° C./min and temperature cycled between 5° C. and 40° C. for 1 cycle(0.1° C./min) prior to holding at 5° C. for approximately 6 hours. Solidmaterials were filtered and dried under vacuum at approximately 40° C.for approximately 21 hours. Isolated material analyzed by XRPD to have aunique crystalline form, designated to be Form 12.

Alternatively, N-methylpyrrolidone:methanol (75:25) was added to BT-11free base in a 42:1 (w/v) ratio to form a slurry by stirring for 1 hourat 50° C. BT-11 dihydrochloride Form 2 was seeded into the mixture(approximately 4% weight). Approximately 2.2 equivalents of HCl wereadded dropwise. An additional approximate 4% weight of BT-11dihydrochloride Form 2 was added. The slurry was stirred at 50° C. forapproximately 18 hours prior to cooling to 5° C. at a rate of 0.1°C./min and holding at 5° C. for approximately 18 hours. Solid materialswere filtered. Isolated material analyzed by XRPD to have a uniquecrystalline form, designated to be Form 12.

Characterization

BT-11 Form 12 was characterized via XRPD (FIG. 37).

Example 14. Crystal Form 13 of BT-11 Form 13 Preparation Methods

DMSO:water (50:50) was added to BT-11 free base in a 59:1 (w/v) ratio toform a slurry by stirring for 1 hour at 50° C. BT-11 dihydrochlorideForm 2 was seeded into the mixture (approximately 4% weight).Approximately 2.2 equivalents of HCl were added in a single aliquot. Anadditional approximate 4% weight of BT-11 dihydrochloride Form 2 wasadded. The slurry was stirred at 50° C. for approximately 18 hours priorto cooling to 5° C. at a rate of 0.1° C./min and holding at 5° C. forapproximately 18 hours. Solid materials were filtered. Isolated materialanalyzed by XRPD to have a unique crystalline form, designated to beForm 13.

Characterization

BT-11 Form 13 was characterized via XRPD (FIG. 38).

Example 15. Pharmacokinetic Analysis Experimental Methods

BT-11 Form 0, Form 1, and Form 2 were orally dosed to male and femalemice (n=4) to provide 8 mg/kg. Plasma was collected at 1, 2, and 4 hourspost-dose. At 4 hours post-dose, colons were excised and washed in PBS.Tissues were then homogenized by Fast-Prep24 instrument. Plasma andtissue homogenate were prepared by acetonitrile-based liquid-liquidextraction. BT-11 concentrations were determined by LC/MS/MS analysisusing a linear calibration curve.

Results

Bioanalytical results of maximum concentration in plasma and colonicconcentrations 4 hours post-dose are presented in Table 20.

TABLE 20 Bioanalytical analysis of BT-11 in plasma and colonic tissue.Form Form 0 Form 1 Form 2 Plasma BT-11 C_(max) (ng/mL) 186 177 41 ColonBT-11 Concentration (ng/mg) 1275 3000 4470

Exemplary Embodiments of the Invention

1. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one 2θ value range selected from 12.4 to12.8, 16.9 to 17.3, 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6, and 28.2to 28.6 degrees in an X-ray powder diffraction pattern obtained using CuK alpha radiation.

2. The crystal form of embodiment 1, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within one or both 2θ value range(s) selected from 12.4 to12.8 and 16.9 to 17.3.

3. The crystal form of any one of embodiments 1-2, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 12.4 to 12.8 degrees.

4. The crystal form of any one of embodiments 1-3, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 16.9 to 17.3 degrees.

5. The crystal form of any one of embodiments 1-4, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, at least three, or each 2θvalue range(s) selected from 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6,and 28.2 to 28.6 degrees.

6. The crystal form of any one of embodiments 1-5, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 2θ value range selected from 12.4 to 12.8, 16.9to 17.3, 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6, and 28.2 to 28.6degrees.

7. The crystal form of any one of embodiments 1-6, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 9.8 to 10.2 degrees.

8. The crystal form of any one of embodiments 1-7, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 14.1 to 14.5 degrees.

9. The crystal form of any one of embodiments 1-8, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 19.2 to 19.6 degrees.

10. The crystal form of any one of embodiments 1-9, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 25.3 to 25.7 degrees.

11. The crystal form of any one of embodiments 1-10, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a Dv50 within a range of 13-55 μm, a Dv10 within a range of 5-28 μm,and a Dv90 within a range of 18-117 μm, with the proviso that the Dv10is a lower value than the Dv50 and the Dv50 is a lower value than theDv90.

12. The crystal form of any one of embodiments 1-11, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 11, 12, 13, 14A, and 14B.

13. The crystal form of any one of embodiments 1-12, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has unit cell dimensions a=12.81 Å, α=90°, b=12.56 Å, β=105.25°, c=9.48Å, γ=90°.

14. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=12.81 Å, α=90°, b=12.56 Å, β=105.25°,c=9.48 Å, γ=90°.

15. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one 2θ value range selected from 10.1 to10.5, 13.8 to 14.2, 16.9 to 17.3, 23.7 to 24.1, 24.3 to 24.7, 27.7 to28.1, and 28.8 to 29.2 degrees in an X-ray powder diffraction patternobtained using Cu K alpha radiation.

16. The crystal form of embodiment 15, wherein thepiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, at least three, or each 2θvalue range(s) selected from 10.1 to 10.5, 16.9 to 17.3, 23.7 to 24.1,and 28.8 to 29.2 degrees. 17. The crystal form of any one of embodiments15-16, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 10.1 to 10.5 degrees.

18. The crystal form of any one of embodiments 15-17, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 16.9 to 17.3 degrees.

19. The crystal form of any one of embodiments 15-18, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 23.7 to 24.1 degrees.

20. The crystal form of any one of embodiments 15-19, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 28.8 to 29.2 degrees.

21. The crystal form of any one of embodiments 15-20, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has peak within at least one, at least two, or three 2θ value range(s)selected from 13.8 to 14.2, 24.3 to 24.7, and 27.7 to 28.1 degrees.

22. The crystal form of any one of embodiments 15-21, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 2θ value range selected from 10.1 to 10.5, 13.8to 14.2, 16.9 to 17.3, 23.7 to 24.1, 24.3 to 24.7, 27.7 to 28.1, and28.8 to 29.2 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

23. The crystal form of any one of embodiments 15-22, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 21A, 21B, 21C, and 21D.

24. The crystal form of any one of embodiments 15-23, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has unit cell dimensions a=9.30 Å, α=71.15°, b=11.78 Å, β=106.99°,c=10.20 Å, γ=108.35°. 25. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=9.30 Å, α=71.15°, b=11.78 Å, β=106.99°,c=10.20 Å, γ=108.35°.

26. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, at least six, or each 2θ value range(s)selected from 5.7 to 6.1, 9.6 to 10.0, 14.0 to 14.4, 19.4 to 19.8, 23.0to 23.4, 24.1 to 24.5, and 27.9 to 28.3 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation.

27. The crystal form of embodiment 26, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 5.7 to 6.1.

28. The crystal form of any one of embodiments 26-27, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 2θ value range selected from 5.7 to 6.1, 9.6 to10.0, 14.0 to 14.4, 19.4 to 19.8, 23.0 to 23.4, 24.1 to 24.5, and 27.9to 28.3 degrees in an X-ray powder diffraction pattern obtained using CuK alpha radiation.

29. The crystal form of any one of embodiments 26-28, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 4A and 4B.

30. The crystal form of any one of claims 26-29, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has unit cell dimensions a=6.97 Å, α=98.26°, b=15.17 Å, β=101.74°,c=9.31 Å, γ=89.23°.

31. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)with the unit cell dimensions a=6.97 Å, α=98.26°, b=15.17 Å, β=101.74°,c=9.31 Å, γ=89.23°.

32. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, or each 2θ value range(s) selected from 12.0to 12.4, 15.0 to 15.4, 15.3 to 15.7, 21.9 to 22.3, 22.2 to 22.6, and28.0 to 28.4 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

33. The crystal form of embodiment 32, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 12.0 to 12.4, 15.0to 15.4, 15.3 to 15.7, 21.9 to 22.3, 22.2 to 22.6, and 28.0 to 28.4degrees in an X-ray powder diffraction pattern obtained using Cu K alpharadiation.

34. The crystal form of any one of embodiments 32-33, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 1.

35. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 6.9 to 7.3, 12.9 to 13.3, 23.4 to 23.8,and 27.3 to 27.7 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

36. The crystal form of embodiment 35, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 6.9 to 7.3, 12.9 to13.3, 23.4 to 23.8, and 27.3 to 27.7 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation.

37. The crystal form of any one of embodiments 35-36, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 25.

38. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 3.5 to 3.9, 7.2 to 7.6, 14.6 to 15.0,and 24.5 to 24.9 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

39. The crystal form of embodiment 38, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 3.5 to 3.9, 7.2 to7.6, 14.6 to 15.0, and 24.5 to 24.9 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation.

40. The crystal form of any one of embodiments 38-39, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 26.

41. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, or each 2θ valuerange(s) selected from 9.2 to 9.6, 26.6 to 27.0, and 27.6 to 28.0degrees in an X-ray powder diffraction pattern obtained using Cu K alpharadiation.

42. The crystal form of embodiment 41, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 9.2 to 9.6, 26.6 to27.0, and 27.6 to 28.0 degrees in an X-ray powder diffraction patternobtained using Cu K alpha radiation.

43. The crystal form of any one of embodiments 41-42, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 28.

44. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 9.9 to 10.3, 12.1 to12.5, 20.5 to 20.9, 25.7 to 26.1, and 26.9 to 27.3 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation.

45. The crystal form of embodiment 44, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 9.9 to 10.3, 12.1 to12.5, 20.5 to 20.9, 25.7 to 26.1, and 26.9 to 27.3 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation.

46. The crystal form of any one of embodiments 44-45, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 30.

47. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 7.4 to 7.8, 13.1 to13.5, 22.1 to 22.5, 23.9 to 24.3, and 25.1 to 25.5 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation.

48. The crystal form of embodiment 47, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 7.4 to 7.8, 13.1 to13.5, 22.1 to 22.5, 23.9 to 24.3, and 25.1 to 25.5 degrees in an X-raypowder diffraction pattern obtained using Cu K alpha radiation.

49. The crystal form of any one of embodiments 47-48, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 31.

50. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 9.9 to 10.3, 12.1 to 12.5, 25.8 to 26.2,and 26.9 to 27.3 degrees in an X-ray powder diffraction pattern obtainedusing Cu K alpha radiation.

51. The crystal form of embodiment 50, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 9.9 to 10.3, 12.1 to12.5, 25.8 to 26.2, and 26.9 to 27.3 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation.

52. The crystal form of any one of embodiments 50-51, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 33.

53. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, at least five, or each 2θ value range(s) selected from 8.8to 9.2, 9.4 to 9.8, 19.1 to 19.5, 23.0 to 23.4, 26.0 to 26.4, and 27.3to 27.7 degrees in an X-ray powder diffraction pattern obtained using CuK alpha radiation.

54. The crystal form of embodiment 53, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 8.8 to 9.2, 9.4 to9.8, 19.1 to 19.5, 23.0 to 23.4, 26.0 to 26.4, and 27.3 to 27.7 degreesin an X-ray powder diffraction pattern obtained using Cu K alpharadiation.

55. The crystal form of any one of embodiments 53-54, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 35.

56. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, atleast four, or each 2θ value range(s) selected from 15.6 to 16.0, 19.4to 19.8, 21.4 to 21.8, 23.3 to 23.7, and 27.1 to 27.5 degrees in anX-ray powder diffraction pattern obtained using Cu K alpha radiation.

57. The crystal form of embodiment 56, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 15.6 to 16.0, 19.4to 19.8, 21.4 to 21.8, 23.3 to 23.7, and 27.1 to 27.5 degrees in anX-ray powder diffraction pattern obtained using Cu K alpha radiation.

58. The crystal form of any one of embodiments 56-57, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in FIG. 36.

59. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, at least three, or each2θ value range(s) selected from 11.6 to 12.0, 18.3 to 18.7, 27.1 to27.5, 28.0 to 28.4 degrees in an X-ray powder diffraction patternobtained using Cu K alpha radiation.

60. The crystal form of embodiment 59, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 11.6 to 12.0, 18.3to 18.7, 27.1 to 27.5, 28.0 to 28.4 degrees in an X-ray powderdiffraction pattern obtained using Cu K alpha radiation.

61. The crystal form of any one of embodiments 59-60, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 37.

62. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one, at least two, or each 2θ valuerange(s) selected from 9.4 to 9.8, 17.0 to 17.4, and 24.5 to 24.9degrees in an X-ray powder diffraction pattern obtained using Cu K alpharadiation.

63. The crystal form of embodiment 62, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 20 value range selected from 9.4 to 9.8, 17.0 to17.4, and 24.5 to 24.9 degrees in an X-ray powder diffraction patternobtained using Cu K alpha radiation.

64. The crystal form of any one of embodiments 62-63, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to the pattern provided in FIG. 38.

65. A composition comprising the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of any one of embodiments 1-64.

66. The composition of embodiment 65, wherein the composition comprisesthe crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in an amount of at least 80% w/w of the of the total amount ofcrystalizedpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)present in the composition.

67. The composition of any one of embodiments 65-66, further comprisinga pharmaceutically acceptable carrier.

68. A method of administeringpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),the method comprising administering the composition of any one ofembodiments 65-68 to a subject.

69. The method of embodiment 68, wherein the subject is a subjectsuffering from a disease.

70. The method of embodiment 69, wherein the disease comprises a chronicinflammatory, immune-mediated, or autoimmune disease.

71. The method of any one of embodiments 69-70, wherein the diseasecomprises a disease of the gastrointestinal tract.

72. The method of any one of embodiments 69-71, wherein the diseasecomprises inflammatory bowel disease. 73. The method of any one ofembodiments 69-71, wherein the disease comprises an eosinophilicdisorder of the gastrointestinal tract.

74. The method of any one of embodiments 69-70, wherein the diseasecomprises a disease of a surface tissue.

75. The method of embodiment 74, wherein the disease comprises any oneor more of psoriasis, cutaneous lupus erythematosus, dermatomyositis,pemphigoid, pemphigus, scleroderma, vasculitis, epidermolysis bullosaacquisita, vitiligo, lichen planus, scleritis, dermatitis or eczema,erythema nodosum, pyoderma gangrenosum, skin fissures, acne,enterocutaneous fistula, skin tags, canker sores, acrodermatitisenteropathica, pyoderma vegetans, leukocytoclastic vasculitis, analfissures, Sweet's syndrome, rosacea, alopecia, keratodermablennorrhagica, rosacea, cold sores, urticaria, actinic keratosis,carbuncle, cellulitis, ichthyosis vulgaris, skin infection, malar rash,photosensitivity, livedo reticularis, livedo reticularis, oral and nasalulcers, purpura, mucositis, hemorrhoids, burn, and sunburn. 76. Themethod of any one of embodiments 69-75, wherein the pharmaceuticalcomposition is administered in an amount effective to treat the disease.

77. A process for preparing the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of any one of embodiments 1-14, the process comprising mixingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in a solvent to form a slurry.

78. The process of embodiment 77, wherein the solvent comprises at leastone of methanol, 2-ethoxyethanol, methyl isobutyl ketone,N-methylpyrrolidone, and dim ethyl sulfoxide.

79. The process of embodiment 77, wherein the solvent is a mixture ofdimethylsulfoxide, methanol, and water.

80. The process of embodiment 79, wherein the mixture ofdimethylsulfoxide, methanol, and water is in a ratio of 45-55:35-45:5-15(dimethylsulfoxide:methanol:water), such as 50:40:10.

81. The process of embodiment 77, wherein the solvent isN-methylpyrrolidone or a mixture of N-methylpyrrolidone and one or moreof methanol, ethanol, 2-propanol, 1-propanol, acetonitrile, acetone,ethyl acetate, and water.

82. The process of embodiment 77, wherein the solvent is selected fromthe group consisting of 2-ethoxyethanol, methyl isobutyl ketone, and amixture of methanol and water.

83. The process of any one of embodiments 77-82, further comprisingisolating crystallized material from the slurry to obtain isolatedmaterial; washing the isolated material with a solvent comprisingmethanol or N-methylpyrrolidone to obtain washed material; and dryingthe washed material to obtain dried material comprising the crystal formofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

84. The process of any one of embodiments 77-83, further comprisingseeding the slurry with the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).

85. The process of any one of embodiments 77-84, further comprisingcyclically heating and cooling the slurry.

86. A process for preparing the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of any one of embodiments 15-25, the process comprising mixingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in a solvent to form a slurry.

87. The process of embodiment 86, wherein solvent comprises at least oneof tetrahydrofuran, 1,4-dioxane, 1-butanol, 1-propanol, 2-methyl THF,butyl acetate, dichloromethane, ethyl acetate, isopropyl alcohol,methanol, methyl ethyl ketone, and tert-butyl methyl ether.

1. A crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)having a peak within at least one 2θ value range selected from 12.4 to12.8, 16.9 to 17.3, 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6, and 28.2to 28.6 degrees in an X-ray powder diffraction pattern obtained using CuK alpha radiation.
 2. The crystal form of claim 1, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within one or both 2θ value range(s) selected from 12.4 to12.8 and 16.9 to 17.3.
 3. The crystal form of claim 1, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 12.4 to 12.8 degrees.
 4. Thecrystal form of claim 1, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within a 2θ value range of 16.9 to 17.3 degrees.
 5. Thecrystal form of claim 2, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within at least one, at least two, at least three, or each 2θvalue range(s) selected from 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6,and 28.2 to 28.6 degrees.
 6. The crystal form of claim 1, wherein thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 2θ value range selected from 12.4 to 12.8, 16.9to 17.3, 20.5 to 20.9, 22.1 to 22.5, 23.2 to 23.6, and 28.2 to 28.6degrees.
 7. The crystal form of claim 6, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 9.8 to 10.2 degrees. 8.The crystal form of claim 6, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 14.1 to 14.5 degrees. 9.The crystal form of claim 6, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 19.2 to 19.6 degrees. 10.The crystal form of claim 6, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has a peak within a 2θ value range of 25.3 to 25.7 degrees. 11.The crystal form of claim 1, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a Dv50 within a range of 13-55 μm, a Dv10 within a range of 5-28 anda Dv90 within a range of 18-117 with the proviso that the Dv10 is alower value than the Dv50 and the Dv50 is a lower value than the Dv90.12. The crystal form of claim 1, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has an X-ray powder diffraction pattern obtained using Cu K alpharadiation substantially similar to any pattern provided in any one ofFIGS. 11, 12, 13, 14A, and 14B.
 13. The crystal form of claim 1, whereinthe crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has unit cell dimensions a=12.81 Å, α=90°, b=12.56 Å, β=105.25°, c=9.48Å, γ=90°.
 14. (canceled)
 15. A composition comprising the crystal formofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of claim
 1. 16. The composition of claim 15, wherein the compositioncomprises the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in an amount of at least 80% w/w of the of the total amount ofcrystalizedpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)present in the composition.
 17. The composition of claim 15, furthercomprising a pharmaceutically acceptable carrier. 18-34. (canceled) 35.A method of administeringpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone),the method comprising administering the composition of claim 15 to asubject.
 36. The method of claim 35, wherein the subject is a subjectsuffering from a disease.
 37. The method of claim 36, wherein thedisease comprises a chronic inflammatory, immune-mediated, or autoimmunedisease.
 38. The method of claim 36, wherein the disease comprises adisease of the gastrointestinal tract.
 39. The method of claim 36,wherein the disease comprises inflammatory bowel disease.
 40. The methodof claim 36, wherein the disease comprises an eosinophilic disorder ofthe gastrointestinal tract.
 41. The method of claim 36, wherein thedisease comprises a disease of a surface tissue.
 42. The method of claim41, wherein the disease comprises any one or more of psoriasis,cutaneous lupus erythematosus, dermatomyositis, pemphigoid, pemphigus,scleroderma, vasculitis, epidermolysis bullosa acquisita, vitiligo,lichen planus, scleritis, dermatitis or eczema, erythema nodosum,pyoderma gangrenosum, skin fissures, acne, enterocutaneous fistula, skintags, canker sores, acrodermatitis enteropathica, pyoderma vegetans,leukocytoclastic vasculitis, anal fissures, Sweet's syndrome, rosacea,alopecia, keratoderma blennorrhagica, rosacea, cold sores, urticaria,actinic keratosis, carbuncle, cellulitis, ichthyosis vulgaris, skininfection, malar rash, photosensitivity, livedo reticularis, livedoreticularis, oral and nasal ulcers, purpura, mucositis, hemorrhoids,burn, and sunburn.
 43. The method of claim 36, wherein thepharmaceutical composition is administered in an amount effective totreat the disease.
 44. A process for preparing the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)of claim 1, the process comprising mixingpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)in a solvent to form a slurry.
 45. (canceled)
 46. The process of claim44, wherein the solvent is a mixture of dimethylsulfoxide, methanol, andwater in a ratio of 45-55:35-45:5-15 (dimethylsulfoxide:methanol:water),and wherein the process further comprises seeding the slurry with thecrystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone).47-52. (canceled)
 53. The crystal form of claim 1, wherein the crystalform ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within both 2θ value range(s) selected from 12.4 to 12.8 and16.9 to 17.3.
 54. The crystal form of claim 53, wherein the crystal formofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)has a peak within each 2θ value range selected from 20.5 to 20.9, 22.1to 22.5, 23.2 to 23.6, and 28.2 to 28.6 degrees.
 55. The crystal form ofclaim 54, wherein the crystal form ofpiperazine-1,4-diylbis((6-(1H-benzo[d]imidazo-2-yl)pyridine-2-yl)methanone)further has: a peak within a 2θ value range of 9.8 to 10.2 degrees; apeak within a 2θ value range of 14.1 to 14.5 degrees; a peak within a 2θvalue range of 19.2 to 19.6 degrees; and a peak within a 2θ value rangeof 25.3 to 25.7 degrees.